Novel Stra6 Polypeptides

ABSTRACT

The present invention is directed to novel polypeptides having sequence similarity to Stra6, a murine retinoic acid responsive protein, and to nucleic acid molecules encoding those polypeptides. Also provided herein are vectors and host cells comprising those nucleic acid sequences, chimeric polypeptide molecules comprising the polypeptides of the present invention fused to heterologous polypeptide sequences, antibodies which bind to the polypeptides of the present invention and to methods for producing the polypeptides of the present invention.

FIELD OF THE INVENTION

The present invention relates generally to the identification andisolation of novel DNA and to the recombinant production of novelpolypeptides having sequence similarity to murine Stra6, a retinoic acidresponsive protein. Some of these molecules were earlier designated as“PRO10282”, but will hereinafter also be referred to as “Stra6”polypeptides.

BACKGROUND OF THE INVENTION Membrane-Bound Proteins

Membrane-bound proteins and receptors can play important roles in, amongother things, the formation, differentiation and maintenance ofmulticellular organisms. The fate of many individual cells, e.g.,proliferation, migration, differentiation, or interaction with othercells, is typically governed by information received from other cellsand/or the immediate environment. This information is often transmittedby secreted polypeptides (for instance, mitogenic factors, survivalfactors, cytotoxic factors, differentiation factors, neuropeptides, andhormones) which are, in turn, received and interpreted by diverse cellreceptors or membrane-bound proteins. Such membrane-bound proteins andcell receptors include, but are not limited to, cytokine receptors,receptor kinases, receptor phosphatases, receptors involved in cell-cellinteractions, and cellular adhesin molecules like selectins andintegrins. For instance, transduction of signals that regulate cellgrowth and differentiation is regulated in part by phosphorylation ofvarious cellular proteins. Protein tyrosine kinases, enzymes thatcatalyze that process, can also act as growth factor receptors. Examplesinclude fibroblast growth factor receptor and nerve growth factorreceptor.

Membrane-bound proteins and receptor molecules have various industrialapplications, including as pharmaceutical and diagnostic agents.Receptor immunoadhesins, for instance, can be employed as therapeuticagents to block receptor-ligand interactions. The membrane-boundproteins can also be employed for screening of potential peptide orsmall molecule inhibitors of the relevant receptor/ligand interaction.

Efforts are being undertaken by both industry and academia to identifynew, native receptor or membrane-bound proteins. Many efforts arefocused on the screening of mammalian recombinant DNA libraries toidentify the coding sequences for novel receptor or membrane-boundproteins.

The Stra6 polypeptides of the present invention share sequence homology(73% identity and 81% similarity) with the murine protein Stra6, whoseexpression is induced by retinoic acid (Bouillet et al., Dev. Biol.170:420-433 [1995]; Bouillet et al., Mech. Dev. 63: 173-186 [1997];Chazaud et al., Dev. Genet. 19: 66-73 [1996]). Since retinoic acid is animportant signaling molecule during vertebrate development, the genesinduced in response to retinoic acid are thought to play a crucial rolein growth and differentiation during embryonic development. The murineStra6 cDNA was isolated from P19 murine embyonal carcinoma cells using asubtractive hybridization approach designed to identify and isolateretinoic acid inducible genes, and does not show similarity withpreviously characterized proteins. It contains highly hydrophobicstretches of amino acid residues that correspond to multipletrans-membrane domains, a characteristic of a membrane integral protein.Based on its expression pattern, Stra6 is thought to play an importantrole in early dorsoventral limb patterning during embyonic developmentand later in the control of endochondral ossification (Chazaud et al.,Dev. Genet. 19: 66-73 [1996]).

The Stra6 polypeptides disclosed herein contain multiple highlyhydrophobic regions that likely constitute trans-membrane domainsindicating that the Stra6 polypeptides are membrane integral proteins.They may function as receptors for an unknown ligand and may be a partof signal transduction pathway with impact on cell growth, developmentor differentiation.

Gene Amplification in Tumor Cells

Malignant tumors (cancers) are the second leading cause of death in theUnited States, after heart disease (Boring et al. CA Cancel J. Clin.43:7 [1993]).

Cancer is characterized by an increase in the number of abnormal, orneoplastic cells derived from a normal tissue which proliferate to forma tumor mass, the invasion of adjacent tissues by these neoplastic tumorcells, and the generation of malignant cells which eventually spread viathe blood or lymphatic system to regional lymph nodes and to distantsites (metastasis). In a cancerous state, a cell proliferates underconditions in which normal cells would not grow. Cancer manifests itselfin a wide variety of forms, characterized by different degrees ofinvasiveness and aggressiveness.

Alteration of gene expression is intimately related to the uncontrolledcell growth and de-differentiation, which are a common feature of allcancers. The genomes of certain well studied tumors have been found toshow decreased expression of recessive genes, usually referred to astumor suppression genes, which would normally function to preventmalignant cell growth, and/or overexpression of certain dominant genes,such as oncogenes, that act to promote malignant growth. Each of thesegenetic changes appears C to be responsible for importing some of thetraits that, in aggregate, represent the full neoplastic phenotype(Hunter, Cell, 64:1129 [1991] and Bishop, Cell, 64:235-248 [1991]).

A well-known mechanism of gene (e.g., oncogene) overexpression in cancercells is gene amplification. This is a process where in the chromosomeof the ancestral cell multiple copies of a particular gene are produced.The process involves unscheduled replication of the region of chromosomecomprising the gene, followed by recombination of the replicatedsegments back into the chromosome (Alitalo et al., Adv. Cancer Res.,47:235-281 [1986]). It is believed that the overexpression of the geneparallels gene amplification, i.e., is proportionate to the number ofcopies made.

Proto-oncogenes that encode growth factors and growth factor receptorshave been identified to play important roles in the pathogenesis ofvarious human malignancies, including breast cancer. For example, it hasbeen found that the human ErbB2 gene (erbB2, also known as her2, orc-erbB-2), which encodes a 185-kd transmembrane glycoprotein receptor(p185^(HER2); HER2) related to the epidermal growth factor receptorEGFR), is overexpressed in about 25% to 30% of human breast cancer(Slamon et al., Science, 235:177-182 [1987]; Slamon et al., Science,244:707-712 [1989]).

It has been reported that gene amplification of a proto-oncogene is anevent typically involved in the more malignant forms of cancer, andcould act as a predictor of clinical outcome (Schwab et al., GenesChromosomes Cancer, 1: 181-193 [1990]; Alitalo et al., supra). Thus,erbB2 overexpression is commonly regarded as a predictor of a poorprognosis. especially in patients with primary disease that involvesaxillary lymph nodes (Slamon et al., [1987] and [1989], supra; Ravdinand Chamness, Gene, 159: 19-27 [1995]; and Hynes and Stern, Biochim.Biophys. Acta, 1198:165-184 [1994]), and has been linked to sensitivityand/or resistance to hormone therapy and chemotherapeutic regimens,including CMF (cyclophosphamide, methotrexate, and fluoruracil) andanthracyclines (Baselga et al., Oncology, 11 (3 Suppl 1):43-48 [1997]).However, despite the association of erbB2 overexpression with poorprognosis, the odds of HER2-positive patients responding clinically totreatment with taxanes were greater than three times those ofHER2-negative patients (Ibid). A recombinant humanized anti-ErbB2(anti-HER2) monoclonal antibody (a humanized version of the murineanti-ErbB2 antibody 4D5, referred to as rhuMAb HER2 or Herceptin™) hasbeen clinically active in patients with ErbB2-overexpressing metastaticbreast cancers that had received extensive prior anticancer therapy.(Baselga et al., J. Clin. Oncol., 14:737-744 [1996]).

In light of the above, there is obvious interest in identifying novelmolecules, methods and compositions which are useful for diagnosing andtreating tumors which are associated with gene amplification.

SUMMARY OF THE INVENTION

A human cDNA clone (designated herein as DNA 148380-2827) has beenidentified that has homology to nucleic acid encoding a murine retinoicacid responsive protein Stra6 and that encodes a 667 amino acid novelpolypeptide, designated in the present application as full-lengthnative-sequence human “PRO10282”. An additional human cDNA clone(designated herein as DNA148389-2827-1) has been identified that encodesa 658 amino acid novel polypeptide (PRO19578), showing significantsequence homology to both murine Stra6 and native sequence humanPRO10282, which is believed to be an alternatively spliced variant ofnative sequence human PRO10282. As discussed hereinafter, in view oftheir homology with murine Stra6, the PRO10282 polypeptides of thepresent invention (including native sequence and variant molecules) willbe also referred to as “Stra6” polypeptides.

In one embodiment, the invention provides an isolated nucleic acidmolecule comprising a nucleotide sequence that encodes a PRO10282polypeptide.

In one aspect, the isolated nucleic acid molecule comprises a nucleotidesequence having at least about 80% sequence identity, preferably atleast about 81% sequence identity, more preferably at least about 82%sequence identity, yet more preferably at least about 83% sequenceidentity, yet more preferably at least about 84% sequence identity, yetmore preferably at least about 85% sequence identity, yet morepreferably at least about 86% sequence identity, yet more preferably atleast about 87% sequence identity, yet more preferably at least about88% sequence identity, yet more preferably at least about 89% sequenceidentity, yet more preferably at least about 90% sequence identity, yetmore preferably at least about 91% sequence identity, yet morepreferably at least about 92% sequence identity, yet more preferably atleast about 93% sequence identity, yet more preferably at least about94% sequence identity, yet more preferably at least about 95% sequenceidentity, yet more preferably at least about 96% sequence identity, yetmore preferably at least about 97% sequence identity, yet morepreferably at least about 98% sequence identity and yet more preferablyat least about 99% sequence identity to (a) a DNA molecule encoding aPRO10282 polypeptide having the sequence of amino acid residues fromabout 1 to about 667, inclusive, of FIG. 2 (SEQ ID NO:2), or (b) a DNAmolecule encoding a PRO19578 polypeptide having the sequence of aminoacid residues from about 1 to about 658, inclusive, of FIG. 7 (SEQ IDNO: 5), or (c) the complement of the DNA molecule of (a) or (b).

In another aspect, the isolated nucleic acid molecule comprises (a) anucleotide sequence encoding a PRO10282 polypeptide having the sequenceof amino acid residues from about 1 to about 667, inclusive, of FIG. 2(SEQ ID NO:2), or (b) a nucleotide sequence encoding a PRO19578polypeptide having the sequence of amino acid residues from about 1 toabout 658, inclusive of FIG. 7 (SEQ ID NO: 5), or (c) the complement ofthe nucleotide sequence of (a) or (b).

In other aspects, the isolated nucleic acid molecule comprises anucleotide sequence having at least about 80% sequence identity,preferably at least about 81% sequence identity, more preferably atleast about 82% sequence identity, yet more preferably at least about83% sequence identity, yet more preferably at least about 84% sequenceidentity, yet more preferably at least about 85% sequence identity, yetmore preferably at least about 86% sequence identity, yet morepreferably at least about 87% sequence identity, yet more preferably atleast about 88% sequence identity, yet more preferably at least about89% sequence identity, yet more preferably at least about 90% sequenceidentity, yet more preferably at least about 91% sequence identity, yetmore preferably at least about 92% sequence identity, yet morepreferably at least about 93% sequence identity, yet more preferably atleast about 94% sequence identity, yet more preferably at least about95% sequence identity, yet more preferably at least about 96% sequenceidentity, yet more preferably at least about 97% sequence identity, yetmore preferably at least about 98% sequence identity and yet morepreferably at least about 99% sequence identity to (a) a DNA moleculehaving the sequence of nucleotides from about 49 to about 2049,inclusive, of FIG. 1 (SEQ ID NO:1), or (b) a DNA molecule having thesequence of nucleotides from about 186 to about 2159, inclusive, of FIG.6 (SEQ ID NO: 4), or (c) the complement of the DNA molecule of (a) or(b).

In another aspect, the isolated nucleic acid molecule comprises (a) thenucleotide sequence of from about 49 to about 2049, inclusive, of FIG. 1(SEQ ID NO:1), or (b) the nucleotide sequence of from about 186 to about2159 of FIG. 6 (SEQ ID NO: 4), or (c) the complement of the nucleotidesequence of (a) or (b).

In a further aspect, the invention concerns an isolated nucleic acidmolecule comprising a nucleotide sequence having at least about 80%sequence identity, preferably at least about 81% sequence identity, morepreferably at least about 82% sequence identity, yet more preferably atleast about 83% sequence identity, yet more preferably at least about84% sequence identity, yet more preferably at least about 85% sequenceidentity, yet more preferably at least about 86% sequence identity, yetmore preferably at least about 87% sequence identity, yet morepreferably at least about 88% sequence identity, yet more preferably atleast about 89% sequence identity, yet more preferably at least about90% sequence identity, yet more preferably at least about 91% sequenceidentity, yet more preferably at least about 92% sequence identity, yetmore preferably at least about 93% sequence identity, yet morepreferably at least about 94% sequence identity, yet more preferably atleast about 95% sequence identity, yet more preferably at least about96% sequence identity, yet more preferably at least about 97% sequenceidentity, yet more preferably at least about 98% sequence identity andyet more preferably at least about 99% sequence identity to (a) a DNAmolecule that encodes the same mature polypeptide encoded by the humanprotein cDNA deposited with the ATCC on Jan. 11, 2000 under ATCC DepositNo. PTA-1181 (DNA 148380-2827), (b) a DNA molecule that encodes the samemature polypeptide encoded by the human protein cDNA deposited with theATCC on Feb. 23, 2000 under ATCC Deposit No. PTA-1402(DNA148389-2827-1), or (c) the complement of the DNA molecule of (a) or(b). In a preferred embodiment, the isolated nucleic acid moleculecomprises (a) a nucleotide sequence encoding the same mature polypeptideencoded by the human protein cDNA deposited with the ATCC on Jan. 11,2000 under ATCC Deposit No. PTA-1181 (DNA 148380-2827), (b) a nucleotidesequence encoding the same mature polypeptide encoded by the humanprotein cDNA deposited with the ATCC on Feb. 23, 2000 under ATCC DepositNo. PTA-1402 (DNA148389-2827-1), or (c) the complement of the nucleotidesequence of (a) or (b).

In another aspect, the invention concerns an isolated nucleic acidmolecule comprising a nucleotide sequence having at least about 80%sequence identity, preferably at least about 81% sequence identity, morepreferably at least about 82% sequence identity, yet more preferably atleast about 83% sequence identity, yet more preferably at least about84% sequence identity, yet more preferably at least about 85% sequenceidentity, yet more preferably at least about 86% sequence identity, yetmore preferably at least about 87% sequence identity, yet morepreferably at least about 88% sequence identity, yet more preferably atleast about 89% sequence identity, yet more preferably at least about90% sequence identity, yet more preferably at least about 91% sequenceidentity, yet more preferably at least about 92% sequence identity, yetmore preferably at least about 93% sequence identity, yet morepreferably at least about 94% sequence identity, yet more preferably atleast about 95% sequence identity, yet more preferably at least about96% sequence identity, yet more preferably at least about 97% sequenceidentity, yet more preferably at least about 98% sequence identity andyet more preferably at least about 99% sequence identity to (a) thefull-length polypeptide coding sequence of the human protein cDNAdeposited with the ATCC on Jan. 11, 2000 under ATCC Deposit No. PTA-1181(DNA 148380-2827), (b) the full-length polypeptide coding sequence ofthe human protein cDNA deposited with the ATCC on Feb. 23, 2000 underATCC No. PTA-1402 (DNA 148389-2827-1), or (c) the complement of thenucleotide sequence of (a) or (b).

In a preferred embodiment, the isolated nucleic acid molecule comprises(a) the full-length polypeptide coding sequence of the DNA depositedwith the ATCC on Jan. 11, 2000 under ATCC Deposit No. PT-1181 (DNA148380-2827), or (b) the full-length polypeptide coding sequence of theDNA deposited with the ATCC on Feb. 23, 2000 under ATCC Deposit No.PTA-1402 (DNA148389-2827-1), or (c) the complement of the nucleotidesequence of (a) or (b).

In another aspect, the invention concerns an isolated nucleic acidmolecule which encodes an active PRO10282 polypeptide as defined belowcomprising a nucleotide sequence that hybridizes to the complement of anucleic acid sequence that encodes amino acids 1 to about 667,inclusive, of FIG. 2 (SEQ ID NO:2), or to the complement of a nucleicacid sequence that encodes amino acids 1 to about 658, inclusive, ofFIG. 7 (SEQ ID NO: 5), wherein the isolated nucleic acid molecule isother than DNA encoding murine Stra6. Preferably, hybridization occursunder high stringency (stringent) hybridization and wash conditions.

In yet another aspect, the invention concerns an isolated nucleic acidmolecule which encodes an active PRO10282 polypeptide as defined belowcomprising a nucleotide sequence that hybridizes to the complement ofthe nucleic acid sequence between about nucleotides 49 and about 2049,inclusive, of FIG. 1 (SEQ ID NO: 1), or to the complement of the nucleicacid sequence between about nucleotides 186 to about 2159, inclusive, orFIG. 6 (SEQ ID NO: 4), wherein the isolated nucleic acid is other thanDNA encoding murine Stra 6. Preferably, hybridization occurs under highstringency (stringent) hybridization and wash conditions.

In a further aspect, the invention concerns an isolated nucleic acidmolecule having at least about 765 nucleotides and which is produced byhybridizing a test DNA molecule under stringent conditions with (a) aDNA molecule encoding a PRO10282 polypeptide having the sequence ofamino acid residues from about 1 to about 667, inclusive, of FIG. 2 (SEQID NO:2), or (b) a DNA molecule encoding a PRO19578 polypeptide havingthe sequence of amino acid residues from about 1 to about 658,inclusive, of FIG. 7 (SEQ ID NO: 5), or (c) the complement of the DNAmolecule of (a) or (b), and, if the test DNA molecule has at least aboutan 80% sequence identity, preferably at least about an 81% sequenceidentity, more preferably at least about an 82% sequence identity, yetmore preferably at least about an 83% sequence identity, yet morepreferably at least about an 84% sequence identity, yet more preferablyat least about an 85% sequence identity, yet more preferably at leastabout an 86% sequence identity, yet more preferably at least about an87% sequence identity, yet more preferably at least about an 88%sequence identity, yet more preferably at least about an 89% sequenceidentity, yet more preferably at least about a 90% sequence identity,yet more preferably at least about a 91% sequence identity, yet morepreferably at least about a 92% sequence identity, yet more preferablyat least about a 93% sequence identity, yet more preferably at leastabout a 94% sequence identity, yet more preferably at least about a 95%sequence identity, yet more preferably at least about a 96% sequenceidentity, yet more preferably at least about a 97% sequence identity,yet more preferably at least about a 98% sequence identity and yet morepreferably at least about a 99% sequence identity to (a), (b) or (c),isolating the test DNA molecule.

In another aspect, the invention concerns an isolated nucleic acidmolecule comprising a nucleotide sequence encoding a polypeptide scoringat least about 80% positives, preferably at least about 81% positives,more preferably at least about 82% positives, yet more preferably atleast about 83% positives, yet more preferably at least about 84%positives, yet more preferably at least about 85% positives, yet morepreferably at least about 86% positives, yet more preferably at leastabout 87% positives, yet more preferably at least about 88% positives,yet more preferably at least about 89% positives, yet more preferably atleast about 90% positives, yet more preferably at least about 91%positives, yet more preferably at least about 92% positives, yet morepreferably at least about 93% positives, yet more preferably at leastabout 94% positives, yet more preferably at least about 95% positives,yet more preferably at least about 96% positives, yet more preferably atleast about 97% positives, yet more preferably at least about 98%positives and yet more preferably at least about 99% positives whencompared with (a) the amino acid sequence of residues about 1 to 667,inclusive, of FIG. 2 (SEQ ID NO:2), or (b) the amino acid sequence ofresidues about 1 to about 658 of FIG. 7 (SEQ ID NO: 5), or (c) thecomplement of the nucleotide sequence of (a) or (b).

Another aspect the invention provides an isolated nucleic acid moleculecomprising a nucleotide sequence encoding a PRO10282 polypeptide whichis either transmembrane domain-deleted or transmembranedomain-inactivated, or is complementary to such encoding nucleotidesequence, wherein the transmembrane domains have been tentativelyidentified as extending from about amino acid position 54 to about aminoacid position 69, about amino acid position 102 to about amino acidposition 119, about amino acid position 148 to about amino acid position166, about amino acid position 207 to about amino acid position 222,about amino acid position 301 to about amino acid position 320, aboutamino acid position 364 to about amino acid position 380, about aminoacid position 431 to about amino acid position 451, about amino acidposition 474 to about amino acid position 489 and about amino acidposition 512 to about amino acid position 531 in the sequence of FIG. 2(SEQ ID NO:2); and about amino acid position 54 to about amino acidposition 71, about amino acid position 93 to about amino acid position11, about amino acid position 140 to about amino acid position 157,about amino acid position 197 to about amino acid position 214, aboutamino acid position 291 to about amino acid position 312, about aminoacid position 356 to about amino acid position 371, about amino acidposition 425 to about amino acid position 481, about amino acid position505 to about amino acid position 522 of FIG. 7 (SEQ ID NO: 5).Therefore, soluble extracellular domains of the herein describedPRO10282 polypeptides are contemplated.

In this regard, another aspect of the present invention is directed toan isolated nucleic acid molecule which comprises a nucleotide sequencehaving at least about 80% sequence identity, preferably at least about81% sequence identity, more preferably at least about 82% sequenceidentity, yet more preferably at least about 83% sequence identity, yetmore preferably at least about 84% sequence identity, yet morepreferably at least about 85% sequence identity, yet more preferably atleast about 86% sequence identity, yet more preferably at least about87% sequence identity, yet more preferably at least about 88% sequenceidentity, yet more preferably at least about 89% sequence identity, yetmore preferably at least about 90% sequence identity, yet morepreferably at least about 91% sequence identity, yet more preferably atleast about 92% sequence identity, yet more preferably at least about93% sequence identity, yet more preferably at least about 94% sequenceidentity, yet more preferably at least about 95% sequence identity, yetmore preferably at least about 96% sequence identity, yet morepreferably at least about 97% sequence identity, yet more preferably atleast about 98% sequence identity and yet more preferably at least about99% sequence identity to (a) a DNA molecule encoding amino acids 1 to Xof FIG. 2 (SEQ ID NO:2), where X is any amino acid from 49 to 59 of FIG.2 (SEQ ID NO:2), or (b) the complement of the DNA molecule of (a). In aspecific aspect, the isolated nucleic acid molecule comprises anucleotide sequence which (a) encodes amino acids 1 to X of FIG. 2 (SEQID NO:2), or of FIG. 7 (SEQ ID NO: 5), where X is any amino acid from 49to 59 of FIG. 2 (SEQ ID NO:2), or (b) is the complement of the DNAmolecule of (a). In a specific aspect, the isolated nucleic acidmolecule comprises a nucleotide sequence which (a) encodes amino acids 1to X of FIG. 2 (SEQ ID NO: 2), or of FIG. 7 (SEQ ID NO: 5), where X isany amino acid from 49 to 59, inclusive, of FIG. 2 (SEQ ID NO: 2), orFIG. 7 (SEQ ID NO: 5), or (b) is the complement of the DNA molecule of(a).

In yet another aspect of the present invention, the isolated nucleicacid molecule (a) encodes a polypeptide scoring at least about 80%positives, preferably at least about 81% positives, more preferably atleast about 82% positives, yet more preferably at least about 83%positives, yet more preferably at least about 84% positives, yet morepreferably at least about 85% positives, yet more preferably at leastabout 86% positives, yet more preferably at least about 87% positives,yet more preferably at least about 88% positives, yet more preferably atleast about 89% positives, yet more preferably at least about 90%positives, yet more preferably at least about 91% positives, yet morepreferably at least about 92% positives, yet more preferably at leastabout 93% positives, yet more preferably at least about 94% positives,yet more preferably at least about 95% positives, yet more preferably atleast about 96% positives, yet more preferably at least about 97%positives, yet more preferably at least about 98% positives and yet morepreferably at least about 99% positives when compared with the aminoacid sequence of residues about 1 to X of FIG. 2 (SEQ ID NO:2), or ofFIG. 7 (SEQ ID NO: 5), where X is any amino acid from 49 to 59 of FIG. 2(SEQ ID NO:2), or (b) is the complement of the DNA molecule of (a).

Another embodiment is directed to fragments of a PRO10282 polypeptidecoding sequence that may find use as, for example, hybridization probesor for encoding fragments of a PRO10282 polypeptide that may optionallyencode a polypeptide comprising a binding site for an anti-PRO10282antibody. Such nucleic acid fragments are usually at least about 20nucleotides in length, preferably at least about 30 nucleotides inlength, more preferably at least about 40 nucleotides in length, yetmore preferably at least about 50 nucleotides in length, yet morepreferably at least about 60 nucleotides in length, yet more preferablyat least about 70 nucleotides in length, yet more preferably at leastabout 80 nucleotides in length, yet more preferably at least about 90nucleotides in length, yet more preferably at least about 100nucleotides in length, yet more preferably at least about 110nucleotides in length, yet more preferably at least about 120nucleotides in length, yet more preferably at least about 130nucleotides in length, yet more preferably at least about 140nucleotides in length, yet more preferably at least about 150nucleotides in length, yet more preferably at least about 160nucleotides in length, yet more preferably at least about 170nucleotides in length, yet more preferably at least about 180nucleotides in length, yet more preferably at least about 190nucleotides in length, yet more preferably at least about 200nucleotides in length, yet more preferably at least about 250nucleotides in length, yet more preferably at least about 300nucleotides in length, yet more preferably at least about 350nucleotides in length, yet more preferably at least about 400nucleotides in length, yet more preferably at least about 450nucleotides in length, yet more preferably at least about 500nucleotides in length, yet more preferably at least about 600nucleotides in length, yet more preferably at least about 700nucleotides in length, yet more preferably at least about 800nucleotides in length, yet more preferably at least about 900nucleotides in length and yet more preferably at least about 1000nucleotides in length, wherein in this context the term “about” meansthe referenced nucleotide sequence length plus or minus 10% of thatreferenced length. The nucleic acid fragments of the present inventionare different from fragments of the native coding sequence of mouseStra6. In a preferred embodiment, the nucleotide sequence fragment isderived from any coding region of the nucleotide sequence shown in FIG.1 (SEQ ID NO:1), or of the nucleotide sequence shown in FIG. 6 (SEQ IDNO: 4). It is noted that novel fragments of a PRO10282polypeptide-encoding nucleotide sequence may be determined in a routinemanner by aligning the PRO10282 polypeptide-encoding nucleotide sequencewith other known nucleotide sequences using any of a number of wellknown sequence alignment programs and determining which PRO10282polypeptide-encoding nucleotide sequence fragment(s) are novel. All ofsuch PRO10282 polypeptide-encoding nucleotide sequences are contemplatedherein and can be determined without undue experimentation. Alsocontemplated are the PRO10282 polypeptide fragments encoded by thesenucleotide molecule fragments, preferably those PRO10282 polypeptidefragments that comprise a binding site for an anti-PRO10282 antibody.

In another embodiment, the invention provides a vector comprising anucleotide sequence encoding PRO10282 or its variants. The vector maycomprise any of the isolated nucleic acid molecules hereinaboveidentified.

A host cell comprising such a vector is also provided. By way ofexample, the host cells may be CHO cells, E. coli, or yeast. A processfor producing PRO10282 polypeptides is further provided and comprisesculturing host cells under conditions suitable for expression ofPRO10282 and recovering PRO10282 from the cell culture.

In another embodiment, the invention provides isolated PRO10282polypeptide encoded by any of the isolated nucleic acid sequenceshereinabove identified.

In a specific aspect, the invention provides isolated native sequencePRO10282 polypeptide, which in certain embodiments, includes an aminoacid sequence comprising residues from about 1 to about 667 of FIG. 2(SEQ ID NO:2), or residues from about 1 to about 658 of FIG. 7 (SEQ IDNO: 5).

In another aspect, the invention concerns an isolated PRO10282polypeptide, comprising an amino acid sequence having at least about 80%sequence identity, preferably at least about 81% sequence identity, morepreferably at least about 82% sequence identity, yet more preferably atleast about 83% sequence identity, yet more preferably at least about84% sequence identity, yet more preferably at least about 85% sequenceidentity, yet more preferably at least about 86% sequence identity, yetmore preferably at least about 87% sequence identity, yet morepreferably at least about 88% sequence identity, yet more preferably atleast about 89% sequence identity, yet more preferably at least about90% sequence identity, yet more preferably at least about 91% sequenceidentity, yet more preferably at least about 92% sequence identity, yetmore preferably at least about 93% sequence identity, yet morepreferably at least about 94% sequence identity, yet more preferably atleast about 95% sequence identity, yet more preferably at least about96% sequence identity, yet more preferably at least about 97% sequenceidentity, yet more preferably at least about 98% sequence identity andyet more preferably at least about 99% sequence identity to the sequenceof amino acid residues from about 1 to about 667, inclusive, of FIG. 2(SEQ ID NO:2), or to the sequence of amino acid residues from about 1 toabout 658, inclusive, of FIG. 7 (SEQ ID NO: 5).

In a further aspect, the invention concerns an isolated PRO10282polypeptide comprising an amino acid sequence having at least about 80%sequence identity, preferably at least about 81% sequence identity, morepreferably at least about 82% sequence identity, yet more preferably atleast about 83% sequence identity, yet more preferably at least about84% sequence identity, yet more preferably at least about 85% sequenceidentity, yet more preferably at least about 86% sequence identity, yetmore preferably at least about 87% sequence identity, yet morepreferably at least about 88% sequence identity, yet more preferably atleast about 89% sequence identity, yet more preferably at least about90% sequence identity, yet more preferably at least about 91% sequenceidentity, yet more preferably at least about 92% sequence identity, yetmore preferably at least about 93% sequence identity, yet morepreferably at least about 94% sequence identity, yet more preferably atleast about 95% sequence identity, yet more preferably at least about96% sequence identity, yet more preferably at least about 97% sequenceidentity, yet more preferably at least about 98% sequence identity andyet more preferably at least about 99% sequence identity to an aminoacid sequence encoded by the human protein cDNA deposited with the ATCCon Jan. 11, 2000 under ATCC Deposit No. PTA-1181 (DNA 148380-2827), orto an amino acid sequence encoded by the human protein cDNA depositedwith the ATCC on Feb. 23, 2000 under ATCC Deposit No. PTA-1402(DNA14389-2827-1). In a preferred embodiment, the isolated PRO10282polypeptide comprises an amino acid sequence encoded by the humanprotein cDNA deposited with the ATCC on Jan. 11, 2000 under ATCC DepositNo. PTA-1181 (DNA 148380-2827), or by the human protein cDNA depositedwith the ATCC on Feb. 23, 2000 under ATCC Deposit No. PTA-1402 (DNA148389-2827-1).

In a further aspect, the invention concerns an isolated PRO10282polypeptide comprising an amino acid sequence scoring at least about 80%positives, preferably at least about 81% positives, more preferably atleast about 82% positives, yet more preferably at least about 83%positives, yet more preferably at least about 84% positives, yet morepreferably at least about 85% positives, yet more preferably at leastabout 86% positives, yet more preferably at least about 87% positives,yet more preferably at least about 88% positives, yet more preferably atleast about 89% positives, yet more preferably at least about 90%positives, yet more preferably at least about 91% positives, yet morepreferably at least about 92% positives, yet more preferably at leastabout 93% positives, yet more preferably at least about 94% positives,yet more preferably at least about 95% positives, yet more preferably atleast about 96% positives, yet more preferably at least about 97%positives, yet more preferably at least about 98% positives and yet morepreferably at least about 99% positives when compared with the aminoacid sequence of residues from about 1 to about 667, inclusive, of FIG.2 (SEQ ID NO:2), or with the amino acid sequence of residues from about1 to about 659, inclusive, of FIG. 7 (SEQ ID NO: 5).

Another aspect the invention provides an isolated PRO10282 polypeptidewhich is either transmembrane domain-deleted or transmembranedomain-inactivated: Processes for producing the same are also hereindescribed, wherein those processes comprise culturing a host cellcomprising a vector which comprises the appropriate encoding nucleicacid molecule under conditions suitable for expression of the PRO10282polypeptide and recovering the PRO10282 polypeptide from the cellculture.

As such, one aspect of the present invention is directed to an isolatedsoluble PRO10282 polypeptide which comprises an amino acid sequencehaving at least about 80% sequence identity, preferably at least about81% sequence identity, more preferably at least about 82% sequenceidentity, yet more preferably at least about 83% sequence identity, yetmore preferably at least about 84% sequence identity, yet morepreferably at least about 85% sequence identity, yet more preferably atleast about 86% sequence identity, yet more preferably at least about87% sequence identity, yet more preferably at least about 88% sequenceidentity, yet more preferably at least about 89% sequence identity, yetmore preferably at least about 90% sequence identity, yet morepreferably at least about 91% sequence identity, yet more preferably atleast about 92% sequence identity, yet more preferably at least about93% sequence identity, yet more preferably at least about 94% sequenceidentity, yet more preferably at least about 95% sequence identity, yetmore preferably at least about 96% sequence identity, yet morepreferably at least about 97% sequence identity, yet more preferably atleast about 98% sequence identity and yet more preferably at least about99% sequence identity to amino acids 1 to X of FIG. 2 (SEQ ID NO:2), orof FIG. 7 (SEQ ID NO: 5), where X is any amino acid from 49 to 59 ofFIG. 2 (SEQ ID NO:2), or of FIG. 7 (SEQ ID NO: 5). In a preferredaspect, the isolated soluble PRO10282 polypeptide comprises amino acids1 to X of FIG. 2 (SEQ ID NO:2), or of FIG. 7 (SEQ ID NO: 5), where X isany amino acid from 49 to 59 of FIG. 2 (SEQ ID NO:2) or of FIG. 7 (SEQID NO: 5).

In yet another aspect of the present invention, the isolated solublePRO10282 polypeptide comprises an amino acid sequence which scores atleast about 80% positives, preferably at least about 81% positives, morepreferably at least about 82% positives, yet more preferably at leastabout 83% positives, yet more preferably at least about 84% positives,yet more preferably at least about 85% positives, yet more preferably atleast about 86% positives, yet more preferably at least about 87%positives, yet more preferably at least about 88% positives, yet morepreferably at least about 89% positives, yet more preferably at leastabout 90% positives, yet more preferably at least about 91% positives,yet more preferably at least about 92% positives, yet more preferably atleast about 93% positives, yet more preferably at least about 94%positives, yet more preferably at least about 95% positives, yet morepreferably at least about 96% positives, yet more preferably at leastabout 97% positives, yet more preferably at least about 98% positivesand yet more preferably at least about 99% positives when compared withthe amino acid sequence of residues about 1 to X of FIG. 2 (SEQ IDNO:2), or of FIG. 7 (SEQ ID NO: 5), where X is any amino acid from 49 to59 of FIG. 2 (SEQ ID NO:2) or of FIG. 7 (SEQ ID NO: 5).

In yet another aspect, the invention concerns an isolated PRO10282polypeptide, comprising the sequence of amino acid residues from about 1to about 667, inclusive, of FIG. 2 (SEQ ID NO:2), or of amino acidresidues from about 1 to about 658, inclusive, of FIG. 7 (SEQ ID NO: 5),or a fragment thereof which is biologically active or sufficient toprovide a binding site for an anti-PRO10282 antibody, wherein theidentification of PRO10282 polypeptide fragments that possess biologicalactivity or provide a binding site for an anti-PRO10282 antibody may beaccomplished in a routine manner using techniques which are well knownin the art. The fragment herein is other than a fragment of a mouseStra6 polypeptide. Preferably, the PRO10282 fragment retains aqualitative biological activity of a native PRO10282 polypeptide.

In a still further aspect, the invention provides a polypeptide producedby (i) hybridizing a test DNA molecule under stringent conditions with(a) a DNA molecule encoding a PRO10282 polypeptide having the sequenceof amino acid residues from about 1 to about 667, inclusive, of FIG. 2(SEQ ID NO:2), or (b) a DNA molecule encoding a PRO19578 polypeptidehaving the sequence of amino acid residues from about 1 to about 658,inclusive, of FIG. 7 (SEQ ID NO: 5), or (c) the complement of the DNAmolecule of (a) or (b), and if the test DNA molecule has at least aboutan 80% sequence identity, preferably at least about an 81% sequenceidentity, more preferably at least about an 82% sequence identity, yetmore preferably at least about an 83% sequence identity, yet morepreferably at least about an 84% sequence identity, yet more preferablyat least about an 85% sequence identity, yet more preferably at leastabout an 86% sequence identity, yet more preferably at least about an87% sequence identity, yet more preferably at least about an 88%sequence identity, yet more preferably at least about an 89% sequenceidentity, yet more preferably at least about a 90% sequence identity,yet more preferably at least about a 91% sequence identity, yet morepreferably at least about a 92% sequence identity, yet more preferablyat least about a 93% sequence identity, yet more preferably at leastabout a 94% sequence identity, yet more preferably at least about a 95%sequence identity, yet more preferably at least about a 96% sequenceidentity, yet more preferably at least about a 97% sequence identity,yet more preferably at least about a 98% sequence identity and yet morepreferably at least about a 99% sequence identity to (a), (b) or (c),(ii) culturing a host cell comprising the test DNA molecule underconditions suitable for expression of the polypeptide, and (iii)recovering the polypeptide from the cell culture.

In another embodiment, the invention provides chimeric moleculescomprising a PRO10282 polypeptide fused to a heterologous polypeptide oramino acid sequence, wherein the PRO10282 polypeptide may comprise anyPRO10282 polypeptide, variant or fragment thereof as hereinbeforedescribed. An example of such a chimeric molecule comprises a PRO1028%polypeptide fused to an epitope tag sequence or a Fc region of animmunoglobulin.

In another embodiment, the invention provides an antibody as definedbelow which specifically binds to a PRO10282 polypeptide as hereinbeforedescribed. Optionally, the antibody is a monoclonal antibody, anantibody fragment or a single chain antibody.

In yet another embodiment, the invention concerns agonists andantagonists of a native PRO10282 polypeptide as defined below. In aparticular embodiment, the agonist or antagonist is an anti-PRO10282antibody or a small molecule.

In a further embodiment, the invention concerns a method of identifyingagonists or antagonists to a PRO10282 polypeptide which comprisecontacting the PRO10282 polypeptide with a candidate molecule andmonitoring a biological activity mediated by said PRO10282 polypeptide.Preferably, the PRO10282 polypeptide is a native PRO10282 polypeptide.

In a still further embodiment, the invention concerns a composition ofmatter comprising a PRO10282 polypeptide, or an agonist or antagonist ofa PRO10282 polypeptide as herein described, or an anti-PRO10282antibody, in combination with a carrier. Optionally, the carrier is apharmaceutically acceptable carrier.

Another embodiment of the present invention is directed to the use of aPRO10282 polypeptide, or an agonist or antagonist thereof as hereindescribed, or an anti-PRO10282 antibody, for the preparation of amedicament useful in the treatment of a condition which is responsive tothe PRO10282 polypeptide, an agonist or antagonist thereof or ananti-PRO10282 antibody.

In a further aspect, the invention concerns an antibody whichspecifically binds to a PRO10282 polypeptide. The antibody preferablyinduces the death of a cell that expresses the PRO10282 polypeptide. Ina particularly preferred embodiment, the cell is a cancer cell thatoverexpresses a PRO10282 polypeptide as compared to a normal cell of thesame tissue type. The antibody preferably is humanized or human, andincludes antibody fragments and single-chain antibodies.

In another aspect, the invention concerns a composition of mattercomprising an antibody specifically binding to a PRO10282 polypeptide.

In yet another aspect, the invention concerns an isolated nucleic acidmolecule encoding an antibody specifically binding to a PRO10282polypeptide, a vector comprising such nucleic acid, and a host cellcomprising such vector. The invention also concerns a method forproducing anti-PRO10282 antibodies.

In a still further embodiment, the invention concerns a method fordetermining the presence of a PRO10282 polypeptide in a sample suspectedof containing such polypeptide by exposing the sample to ananti-PRO10282 antibody (or another antagonist of PRO10282), anddetermining the binding of such antibody (or antagonist) to the PRO10282polypeptide in the sample. In a particularly important embodiment, thesample is from a cancer cell.

In a different embodiment, the invention concerns a method of diagnosingtumor in a mammal, comprising detecting the level of expression of agene encoding a PRO10282 polypeptide (a) in a test sample of tissueobtained from the mammal, and (b) in a control sample of known normaltissue cells of the same cell type. Higher expression level in the testsample, as compared to the control sample, indicates the presence oftumor in the mammal from which the test tissue cells were taken.

According to another method of the invention, tumor is diagnosed in amammal by (a) contacting an anti-PRO10282 antibody with a test sample oftissue cells obtained from the mammal, and (b) detecting the formationof a complex between the antibody and a PRO10282 polypeptide in the testsample. Formation of a complex is indicative of the presence of a tumorin the mammal.

In another aspect, the invention concerns a cancer diagnostic kitcomprising an anti-PRO10282 antibody (or a different antagonist ofPRO10282) and a carrier in suitable packaging Optionally, the kit alsocontains instructions for using the antibody or other antagonist todetect the presence of a PRO10282 polypeptide in a sample.

In yet another aspect, the invention concerns a method of inhibiting thegrowth of tumor cells by exposing tumor cells that express a PRO10282polypeptide to an effective amount of an agent that inhibits abiological activity of the PRO10282 polypeptide, thereby inhibiting thegrowth of tumor cells. The agent may, for example, be an anti-PRO10282antibody or another antagonist of PRO10282. The tumor cells may befurther exposed to conventional tumor treatments, such as radiationtreatment, treatment with a cytotoxic agent and/or chemotherapeuticagent, etc.

In a different aspect, the invention concerns a method of inhibiting thegrowth of tumor cells by exposing tumor cells that express a PRO10282polypeptide to an effective amount of an agent that inhibits theexpression of the polypeptide, thereby inhibiting tumor growth.

The invention further concerns analogous methods to prevent or slow down(lessen) the development, growth, or spread of tumor characterized bythe overexpression of a PRO10282 polypeptide. Such treatment maydirectly decrease the pathology of tumor cells, or render the tumorcells more susceptible to treatment by other therapeutic agents, e.g.,radiation and/or chemotherapy. The invention specifically includesmethods that control abnormal or otherwise metastasis, interference withthe normal functioning of neighboring cells, release of cytokines orother secretory products at abnormal levels, suppression or aggravationof inflammatory or immunological response, etc.

The invention further concerns articles of manufacture comprising acontainer, a label on a container, and a composition comprising anactive agent, where the composition is effective for inhibiting tumorgrowth and the label on the container indicates that the composition iseffective for treating a condition characterized by overexpression of aPRO10282 polypeptide in tumor cells.

In a still further aspect, the invention concerns a method ofidentifying a compound that inhibits the biological or immunologicalactivity of a PRO10282 polypeptide, by contacting a candidate compoundwith a PRO10282 polypeptide under conditions and for a time sufficientto allow the two components to interact, and determining whether abiological or immunological activity of the PRO10282 polypeptide isinhibited.

In yet another aspect, the invention concerns a method of identifying acompound that inhibits an activity of a PRO10282 polypeptide, comprisingthe following steps: (a) contacting cells and a candidate compound to bescreened in the presence of a PRO10282 polypeptide under conditionssuitable for the induction of a cellular response normally induced by aPRO10282 polypeptide and (b) determining the induction of said cellularresponse to determine if the test compound is an effective antagonist.The lack of induction of a cellular response indicates that the testcompound is an effective antagonist.

The invention further concerns a method for identifying a compound thatinhibits the expression of a PRO10282 polypeptide in cells expressingit, by contacting the cells with a candidate compound and determiningwhether the expression is inhibited.

The invention specifically concerns PRO10282 agonists and antagonistsidentified by the screening methods herein. Such agonists andantagonists include, without limitation, anti-PRO10282 antibodies,polypeptides, peptides, and small organic molecules with agonist orantagonist properties.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the nucleotide sequence (SEQ ID NO:1) of a cDNA containinga nucleotide sequence (nucleotides 1-2732) encoding native sequencePRO10282, wherein the nucleotide sequence (SEQ ID NO:1) is a clonedesignated herein as “DNA148380-2827.” Also presented in bold font andunderlined are the positions of the respective start and stop codons.

FIG. 2 shows the amino acid sequence (SEQ ID NO:2) of a native sequencePRO10282 polypeptide as derived from the coding sequence of SEQ ID NO:1.Also shown are the approximate locations of various other importantpolypeptide domains.

FIGS. 3A-D show hypothetical exemplifications for using the belowdescribed method to determine % amino acid sequence identity (FIGS.3A-B) and % nucleic acid sequence identity (FIGS. 3C-D) using theALIGN-2 sequence comparison computer program, wherein “PRO” representsthe amino acid sequence of a hypothetical PRO10282 polypeptide ofinterest, “Comparison Protein” represents the amino acid sequence of apolypeptide against which the “PRO” polypeptide of interest is beingcompared, “PRO-DNA” represents a hypothetical PRO10282-encoding nucleicacid sequence of interest, “Comparison DNA” represents the nucleotidesequence of a nucleic acid molecule against which the “PRO-DNA” nucleicacid molecule of interest is being compared, “X, “Y” and “Z” eachrepresent different hypothetical amino acid residues and “N”, “L” and“V” each represent different hypothetical nucleotides.

FIGS. 4A-Q provide the complete source code for the ALIGN-2 sequencecomparison computer program. This source code may be routinely compiledfor use on a UNIX operating system to provide the ALIGN-2 sequencecomparison computer program.

FIG. 5 shows a nucleotide sequence designated herein as DNA100038 (SEQID NO:3).

FIG. 6 shows the nucleotide sequence (SEQ ID NO: 4) of a cDNA containinga nucleotide sequence (nucleotides 1-2778) encoding a native sequencehuman Stra6 polypeptide variant, wherein the nucleotide sequence (SEQ IDNO:4) is a clone designated herein as “DNA148389-2827-1.” Also presentedin bold font and underlined are the positions of the respective startand stop codons.

FIG. 7 shows the amino acid sequence (SEQ ID NO:5) of a native sequencehuman Stra6 polypeptide variant as derived from the coding sequence ofSEQ ID NO:4. Also shown are the approximate locations of various otherimportant polypeptide domains.

FIG. 8 is a schematic representation of mouse Stra6, and the human Stra6protein encoded by DNA148380-2827 (native human PRO10282).

FIG. 9 shows the hydrophobicity plot of the native sequence human Stra6protein encoded by DNA 148380-2827 (native human PRO10282).

FIG. 10 shows the relative RNA expression profile for the nativesequence human Stra 6 protein encoded by DNA 148380-2827 in variousnormal human tissues.

FIG. 11 shows the RNA fold expression for the native sequence humanStra6 protein encoded by DNA 148380-2827 in human colon tumor tissuerelative to RNA expression in normal mucose from the same patientassayed by quantitative PCR. The data are from one experiment done intriplicate. The experiment was repeated at least twice with a differentset of PCR primers.

FIG. 12A shows the RNA expression for the native sequence human Stra6protein encoded by DNA 148380-2827 in human colon tissue relative to RNAexpression in normal mucose from the same patient, using thehousekeeping gene, GAPDH as a control.

FIG. 12B shows the localization of Stra6 to the epithelial tumor cellsin a colon adenocarcinoma by in situ hybridization.

FIG. 13 shows the RNA expression for the native human Stra6 proteinencoded by DNA148380-2827 in human breast, kidney, colon and lung tumorcell lines, relative to corresponding normal cell lines.

FIG. 14 illustrates the expression of peptide fragments derived from thenative sequence human Stra 6 protein encoded by DNA 148380-2827 in E.coli.

FIG. 15 illustrates Stra6 RNA expression in human colon carcinoma cellsin the presence and absence of all-trans-retinoic acid (ATRA) and9-cis-retinoic acid (9cRA), respectively.

FIG. 16 In situ hybridization for Stra6 in tumor sections. Darkfieldimages demonstrating silver grains (A, C, E, G) are shown withcorresponding hematoxylin/eosin-stained brightfield images (B, D, F, H).Moderate densities of silver grains overlie tumor cells but not a bloodvessel in a malignant melanoma (A, B). Neoplastic epithelium in anendometrial adenocarcinoma is moderately labeled whereas tumor stroma isnegative (C, D). Blastemal regions in a Wilm's tumor display highexpression levels whereas tumor stroma is negative (E, F). Apheochromocytoma shows very high Stra6 mRNA expression while adjacentnormal adrenal cortex is negative (G, H). Scale bars=100 microns.

FIG. 17 (A) Induction of Stra6 mRNA expression in response to 9-cis-RAor all-trans-RA in C57MG/Parent and C57MG/Wnt-1 cells. (B) Induction ofStra6 mRNA expression in C57MG/Parent cells in response to Wnt-3Aconditioned media and 9-cis-RA. (C) Induction of Stra6 mRNA expressionafter retinoic acid treatment in HCT116 and WiDr colon adenocarcinomacells. (D) Darkfield images demonstrating Stra6 expression by in situhybridization in HCT116 cells before (top panel) and after (lower panel)treatment with retinoic acid. (E) Stra6 protein expression in WiDr cellsbefore (−RA) and after (+RA) treatment with retinoic acid as visualizedby Western blot with a monoclonal antibody directed against human Stra6peptide B. (F) Stra6 membrane localization in WiDr cells untreated (leftpanel) or treated (right panel) with retinoic acid. Immunohistochemistrywas performed with an anti-human Stra6 peptide B monoclonal hybridomaculture supernatant (clone 12F4.2H9.1D5). For the experiments shown inA-F, cells were treated with retinoic acid for 48 hours. Stra6 productsobtained after completion of the quantitative PCR reactions (40 cycleseach) are shown below each graph A-C.

FIG. 18 (A) Wnt-1 induces RARγ-1 expression. Protein-equivalent amountsof whole cell lysate from tetracycline repressible C57MG/Wnt-1 cells inthe absence of tetracycline for 0, 24, 48, or 72 hours, were subjectedto SDS-PAGE and immunoblotted for RARγ-1 and ERK2. (B) RARγ-1 mRNAexpression in hyperplastic mammary glands and mammary gland tumors fromWnt-1 transgenic mice. mRNA expression was determined by quantitativeRT-PCR and the data are expressed as fold expression relative to mRNAexpression in wild-type mammary glands.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 1. Definitions

The terms “PRO10282 polypeptide”, “PRO10282 protein”, “PRO10282”, “Stra6polypeptide”, “Stra6 protein” and “Stra6” are used interchangeably, andencompass native sequence PRO10282 (Stra6) and PRO10282 (Stra6)polypeptide variants (which are further defined herein). The PRO10282(Stra6) polypeptide may be isolated from a variety of sources, such asfrom human tissue types or from another source, or prepared byrecombinant and/or synthetic methods.

A “native sequence PRO10282” or “native sequence Stra6” comprises apolypeptide having the same amino acid sequence as a PRO10282 derivedfrom nature. Such native sequence PRO10282 (Stra6) can be isolated fromnature or can be produced by recombinant and/or synthetic means. Theterm “native sequence PRO10282” or “native sequence Stra6” specificallyencompasses naturally-occurring truncated or secreted forms (e.g., anextracellular domain sequence), naturally-occurring variant forms (e.g.,alternatively spliced forms) and naturally-occurring allelic variants ofthe PRO10282. In one embodiment of the invention, the native sequencePRO10282 is a mature or full-length native sequence PRO10282 comprisingamino acids 1 to 667 of FIG. 2 (SEQ ID NO:2). In another embodiment ofthe invention, the native sequence PRO10282 polypeptide is a mature orfull-length PRO19578 polypeptide comprising amino acids 1 to 658 of SEQID NO: 5, which is believed to be an alternatively spliced form of thenative sequence PRO10282 polypeptide of SEQ ID NO: 2. Also, while thePRO10282 polypeptides disclosed in FIG. 2 (SEQ ID NO:2) and in FIG. 7,SEQ ID NO: 5 are shown to begin with the methionine residue designatedherein as amino acid position 1, it is conceivable and possible thatanother methionine residue located either upstream or downstream fromamino acid position 1 in FIG. 2 (SEQ ID NO:2) or in FIG. 7 (SEQ ID NO:5) may be employed as the starting amino acid residue for the PRO10282polypeptide.

The PRO10282 (Stra6) polypeptide “extracellular domain” or “ECD” refersto a form of the PRO10282 (Stra6) polypeptide which is essentially freeof the transmembrane and cytoplasmic domains. Ordinarily, a PRO10282polypeptide ECD will have less than about 1% of such transmembraneand/or cytoplasmic domains and preferably, will have less than about0.5% of such domains. It will be understood that any transmembranedomain(s) identified for the PRO10282 polypeptides of the presentinvention are identified pursuant to criteria routinely employed in theart for identifying that type of hydrophobic domain. The exactboundaries of a transmembrane domain may vary but most likely by no morethan about 5 amino acids at either end of the domain as initiallyidentified. As such, in one embodiment of the present invention, theextracellular domain of a PRO10282 polypeptide comprises amino acids 1to X, wherein X is any amino acid from amino acid 49 to 59 of FIG. 2(SEQ ID NO:2) or of FIG. 7 (SEQ ID NO: 5).

“PRO10282 variant polypeptide” or “Stra6 variant polypeptide”, whichterms are used interchangeably, means an active PRO10282 (Stra6)polypeptide as defined below having at least about 80% amino acidsequence identity with the amino acid sequence of (a) residues 1 to 667of the PRO10282 polypeptide shown in FIG. 2 (SEQ ID NO:2), or residues 1to 658 of FIG. 7 (SEQ ID NO: 5), (b) 1 to X of FIG. 2 (SEQ ID NO:2) orFIG. 7 (SEQ ID NO: 5), wherein X is any amino acid from amino acid 49 toamino acid 59 of FIG. 2 (SEQ ID NO:2) or of FIG. 7 (SEQ ID NO: 5), or(c) another specifically derived fragment of the amino acid sequenceshown in FIG. 2 (SEQ ID NO:2), or FIG. 7 (SEQ ID NO: 5). Such PRO10282(Stra6) variant polypeptides include, for instance, PRO10282 (Stra6)polypeptides wherein one or more amino acid residues are added, ordeleted, at the N- and/or C-terminus, as well as within one or moreinternal domains, of the sequence of FIG. 2 (SEQ ID NO:2) or FIG. 7 (SEQID NO: 5). Ordinarily, a PRO10282 variant polypeptide will have at leastabout 80% amino acid sequence identity, more preferably at least about81% amino acid sequence identity, more preferably at least about 82%amino acid sequence identity, more preferably at least about 83% aminoacid sequence identity, more preferably at least about 84% amino acidsequence identity, more preferably at least about 85% amino acidsequence identity, more preferably at least about 86% amino acidsequence identity, more preferably at least about 87% amino acidsequence identity, more preferably at least about 88% amino acidsequence identity, more preferably at least about 89% amino acidsequence identity, more preferably at least about 90% amino acidsequence identity, more preferably at least about 91% amino acidsequence identity, more preferably at least about 92% amino acidsequence identity, more preferably at least about 93% amino acidsequence identity, more preferably at least about 94% amino acidsequence identity, more preferably at least about 95% amino acidsequence identity, more preferably at least about 96% amino acidsequence identity, more preferably at least about 97% amino acidsequence identity, more preferably at least about 98% amino acidsequence identity and yet more preferably at least about 99% amino acidsequence identity with (a) residues 1 to 667 of the PRO10282 polypeptideshown in FIG. 2 (SEQ ID NO:2) or residues 1 to 658 of the PRO19578polypeptide of FIG. 7 (SEQ ID NO: 5), (b) 1 to X of FIG. 2 (SEQ ID NO:2)or FIG. 7 (SEQ ID NO: 5), wherein X is any amino acid from amino acid 49to amino acid 59 of FIG. 2 (SEQ ID NO:2) or FIG. 7 (SEQ ID NO: 5), or(c) another specifically derived fragment of the amino acid sequenceshown in FIG. 2 (SEQ ID NO:2) or FIG. 7 (SEQ ID NO: 5). PRO10282 variantpolypeptides do not encompass the native PRO10282 polypeptide sequence.Ordinarily, PRO10282 variant polypeptides are at least about 10 aminoacids in length, often at least about 20 amino acids in length, moreoften at least about 30 amino acids in length, more often at least about40 amino acids in length, more often at least about 50 amino acids inlength, more often at least about 60 amino acids in length, more oftenat least about 70 amino acids in length, more often at least about 80amino acids in length, more often at least about 90 amino acids inlength, more often at least about 100 amino acids in length, more oftenat least about 150 amino acids in length, more often at least about 200amino acids in length, more often at least about 250 amino acids inlength, more often at least about 300 amino acids in length, or more,and are different from fragments of the murine Stra6 sequence, asdisclosed in Bouillet et al., Mechanisms of Development 63, 173-186(1997).

“Percent (%) amino acid sequence identity” with respect to the PRO10282(Stra6) polypeptide sequences identified herein is defined as thepercentage of amino acid residues in a candidate sequence that areidentical with the amino acid residues in a PRO10282 sequence, afteraligning the sequences and introducing gaps, if necessary, to achievethe maximum percent sequence identity, and not considering anyconservative substitutions as part of the sequence identity. Alignmentfor purposes of determining percent amino acid sequence identity can beachieved in various ways that are within the skill in the art, forinstance, using publicly available computer software such as BLAST,BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software. Those skilled inthe art can determine appropriate parameters for measuring alignment,including any algorithms needed to achieve maximal alignment over thefull-length of the sequences being compared. For purposes herein,however, % amino acid sequence identity values are obtained as describedbelow by using the sequence comparison computer program ALIGN-2, whereinthe complete source code for the ALIGN-2 program is provided in FIGS.4A-Q. The ALIGN-2 sequence comparison computer program was authored byGenentech, Inc. and the source code shown in FIGS. 4A-Q has been filedwith user documentation in the U.S. Copyright Office, Washington D.C.,20559, where it is registered under U.S. Copyright Registration No.TXU510087. The ALIGN-2 program is publicly available through Genentech,Inc., South San Francisco, Calif. or may be compiled from the sourcecode provided in FIGS. 4A-Q. The ALIGN-2 program should be compiled foruse on a UNIX operating system, preferably digital UNIX V4.0D. Allsequence comparison parameters are set by the ALIGN-2 program and do notvary.

For purposes herein, the % amino acid sequence identity of a given aminoacid sequence A to, with, or against a given amino acid sequence B(which can alternatively be phrased as a given amino acid sequence Athat has or comprises a certain % amino acid sequence identity to, with,or against a given amino acid sequence B) is calculated as follows:

100 times the fraction X/Y

where X is the number of amino acid residues scored as identical matchesby the sequence alignment program ALIGN-2 in that program's alignment ofA and B, and where Y is the total number of amino acid residues in B. Itwill be appreciated that where the length of amino acid sequence A isnot equal to the length of amino acid sequence B, the % amino acidsequence identity of A to B will not equal the % amino acid sequenceidentity of B to A. As examples of % amino acid sequence identitycalculations, FIGS. 3A-B demonstrate how to calculate the % amino acidsequence identity of the amino acid sequence designated “ComparisonProtein” to the amino acid sequence designated “PRO”.

Unless specifically stated otherwise, all % amino acid sequence identityvalues used herein are obtained as described above using the ALIGN-2sequence comparison computer program. However, % amino acid sequenceidentity may also be determined using the sequence comparison programNCBI-BLAST2 (Altschul et al., Nucleic Acids Res. 25:3389-3402 (1997)).The NCBI-BLAST2 sequence comparison program may be downloaded fromhttp://www.ncbi.nlm.nih.gov. NCBI-BLAST2 uses several search parameters,wherein all of those search parameters are set to default valuesincluding, for example, unmask=yes, strand=all, expected occurrences=10,minimum low complexity length=15/5, multi-pass e-value=0.01, constantfor multi-pass=25, dropoff for final gapped alignment=25 and scoringmatrix=BLOSUM62.

In situations where NCBI-BLAST2 is employed for amino acid sequencecomparisons, the % amino acid sequence identity of a given amino acidsequence A to, with, or against a given amino acid sequence B (which canalternatively be phrased as a given amino acid sequence A that has orcomprises a certain % amino acid sequence identity to, with, or againsta given amino acid sequence B) is calculated as follows:

100 times the fraction X/Y

where X is the number of amino acid residues scored as identical matchesby the sequence alignment program NCBI-BLAST2 in that program'salignment of A and B, and where Y is the total number of amino acidresidues in B. It will be appreciated that where the length of aminoacid sequence A is not equal to the length of amino acid sequence B, the% amino acid sequence identity of A to B will not equal the % amino acidsequence identity of B to A.

“PRO10282 (Stra6) variant polynucleotide” or “PRO10282 (Stra6) variantnucleic acid sequence” means a nucleic acid molecule which encodes anactive PRO10282 polypeptide as defined below and which has at leastabout 80% nucleic acid sequence identity with either (a) a nucleic acidsequence which encodes residues 1 to 667 of the PRO10282 polypeptideshown in FIG. 2 (SEQ ID NO:2), or residues 1 to 658 of FIG. 7 (SEQ IDNO: 5), (b) a nucleic acid sequence which encodes amino acids 1 to X ofFIG. 2 (SEQ ID NO:2) or FIG. 7 (SEQ ID NO: 5), wherein X is any aminoacid from amino acid 49 to amino acid 59 of FIG. 2 (SEQ ID NO:2) or ofFIG. 7 (SEQ ID NO: 5), or (c) a nucleic acid sequence which encodesanother specifically derived fragment of the amino acid sequence shownin FIG. 2 (SEQ ID NO:2) or in FIG. 7 (SEQ ID NO: 5). Ordinarily, aPRO10282 variant polynucleotide will have at least about 80% nucleicacid sequence identity, more preferably at least about 81% nucleic acidsequence identity, more preferably at least about 82% nucleic acidsequence identity, more preferably at least about 83% nucleic acidsequence identity, more preferably at least about 84% nucleic acidsequence identity, more preferably at least about 85% nucleic acidsequence identity, more preferably at least about 86% nucleic acidsequence identity, more preferably at least about 87% nucleic acidsequence identity, more preferably at least about 88% nucleic acidsequence identity, more preferably at least about 89% nucleic acidsequence identity, more preferably at least about 90% nucleic acidsequence identity, more preferably at least about 91% nucleic acidsequence identity, more preferably at least about 92% nucleic acidsequence identity, more preferably at least about 93% nucleic acidsequence identity, more preferably at least about 94% nucleic acidsequence identity, more preferably at least about 95% nucleic acidsequence identity, more preferably at least about 96% nucleic acidsequence identity, more preferably at least about 97% nucleic acidsequence identity, more preferably at least about 98% nucleic acidsequence identity and yet more preferably at least about 99% nucleicacid sequence identity with either (a) a nucleic acid sequence whichencodes residues 1 to 667 of the PRO10282 polypeptide shown in FIG. 2(SEQ ID NO:2), or residues 1 to 658 of the PRO19678 polypeptide shown inFIG. 7 (SEQ ID NO: 5), (b) a nucleic acid sequence which encodes aminoacids 1 to X of FIG. 2 (SEQ ID NO:2) or of FIG. 7 (SEQ ID NO: 5),wherein X is any amino acid from amino acid 49 to amino acid 59 of FIG.2 (SEQ ID NO:2) or FIG. 7 (SEQ ID NO: 5), or (c) a nucleic acid sequencewhich encodes another specifically derived fragment of the amino acidsequence shown in FIG. 2 (SEQ ID NO:2) or in FIG. 7 (SEQ ID NO: 5).PRO10282 polynucleotide variants do not encompass the native PRO10282nucleotide sequence, or the native PRO19578 nucleotide sequence.

Ordinarily, PRO10282 (Stra6) variant polynucleotides are at least about30 nucleotides in length, often at least about 60 nucleotides in length,more often at least about 90 nucleotides in length, more often at leastabout 120 nucleotides in length, more often at least about 150nucleotides in length, more often at least about 180 nucleotides inlength, more often at least about 210 nucleotides in length, more oftenat least about 240 nucleotides in length, more often at least about 270nucleotides in length, more often at least about 300 nucleotides inlength, more often at least about 450 nucleotides in length, more oftenat least about 600 nucleotides in length, more often at least about 900nucleotides in length, or more, and specifically exclude polynucleotidesthat are included in the nucleotide sequence of murine Stra6, asdisclosed in Bouillet et al., Mechanisms of Development 63, 173-186(1997).

“Percent (%) nucleic acid sequence identity” with respect to thePRO10282 (Stra6) polypeptide-encoding nucleic acid sequences identifiedherein is defined as the percentage of nucleotides in a candidatesequence that are identical with the nucleotides in a PRO10282polypeptide-encoding nucleic acid sequence, after aligning the sequencesand introducing gaps, if necessary, to achieve the maximum percentsequence identity. Alignment for purposes of determining percent nucleicacid sequence identity can be achieved in various ways that are withinthe skill in the art, for instance, using publicly available computersoftware such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR)software. Those skilled in the art can determine appropriate parametersfor measuring alignment, including any algorithms needed to achievemaximal alignment over the full-length of the sequences being compared.For purposes herein, however, % nucleic acid sequence identity valuesare obtained as described below by using the sequence comparisoncomputer program ALIGN-2, wherein the complete source code for theALIGN-2 program is provided in FIGS. 4A-Q. The ALIGN-2 sequencecomparison computer program was authored by Genentech, Inc. and thesource code shown in FIGS. 4A-Q has been filed with user documentationin the U.S. Copyright Office, Washington D.C., 20559, where it isregistered under U.S. Copyright Registration No. TXU510087. The ALIGN-2program is publicly available through Genentech, Inc., South SanFrancisco, Calif. or may be compiled from the source code provided inFIGS. 4A-Q. The ALIGN-2 program should be compiled for use on a UNIXoperating system, preferably digital UNIX V4.0D. All sequence comparisonparameters are set by the ALIGN-2 program and do not vary.

For purposes herein, the % nucleic acid sequence identity of a givennucleic acid sequence C to, with, or against a given nucleic acidsequence D (which can alternatively be phrased as a given nucleic acidsequence C that has or comprises a certain % nucleic acid sequenceidentity to, with, or against a given nucleic acid sequence D) iscalculated as follows:

100 times the fraction W/Z

where W is the number of nucleotides scored as identical matches by thesequence alignment program ALIGN-2 in that program's alignment of C andD, and where Z is the total number of nucleotides in D. It will beappreciated that where the length of nucleic acid sequence C is notequal to the length of nucleic acid sequence D, the % nucleic acidsequence identity of C to D will not equal the % nucleic acid sequenceidentity of D to C. As examples of % nucleic acid sequence identitycalculations, FIGS. 3C-D demonstrate how to calculate the % nucleic acidsequence identity of the nucleic acid sequence designated “ComparisonDNA” to the nucleic acid sequence designated “PRO-DNA”.

Unless specifically stated otherwise, all % nucleic acid sequenceidentity values used herein are obtained as described above using theALIGN-2 sequence comparison computer program. However, % nucleic acidsequence identity may also be determined using the sequence comparisonprogram NCBI-BLAST2 (Altschul et al., Nucleic Acids Res. 25:3389-3402(1997)). The NCBI-BLAST2 sequence comparison program may be downloadedfrom http://www.ncbi.nlm.nih.gov. NCBI-BLAST2 uses several searchparameters, wherein all of those search parameters are set to defaultvalues including, for example, unmask=yes, strand=all, expectedoccurrences=10, minimum low complexity length=15/5, multi-passe-value=0.01, constant for multi-pass=25, dropoff for final gappedalignment=25 and scoring matrix=BLOSUM62.

In situations where NCBI-BLAST2 is employed for sequence comparisons,the % nucleic acid sequence identity of a given nucleic acid sequence Cto, with, or against a given nucleic acid sequence D (which canalternatively be phrased as a given nucleic acid sequence C that has orcomprises a certain % nucleic acid sequence identity to, with, oragainst a given nucleic acid sequence D) is calculated as follows:

100 times the fraction W/Z

where W is the number of nucleotides scored as identical matches by thesequence alignment program NCBI-BLAST2 in that program's alignment of Cand D, and where Z is the total number of nucleotides in D. It will beappreciated that where the length of nucleic acid sequence C is notequal to the length of nucleic acid sequence D, the % nucleic acidsequence identity of C to D will not equal the % nucleic acid sequenceidentity of D to C.

In other embodiments, PRO10282 (Stra6) variant polynucleotides arenucleic acid molecules that encode an active PRO10282 polypeptide andwhich are capable of hybridizing, preferably under stringenthybridization and wash conditions, to nucleotide sequences encoding thefull-length PRO10282 polypeptides shown in FIG. 2 (SEQ ID NO:2) or FIG.7 (SEQ ID NO: 5). PRO10282 variant polypeptides may be those that areencoded by a PRO10282 variant polynucleotide.

The term “positives”, in the context of the amino acid sequence identitycomparisons performed as described above, includes amino acid residuesin the sequences compared that are not only identical, but also thosethat have similar properties. Amino acid residues that score a positivevalue to an amino acid residue of interest are those that are eitheridentical to the amino acid residue of interest or are a preferredsubstitution (as defined in Table 1 below) of the amino acid residue ofinterest.

For purposes herein, the % value of positives of a given amino acidsequence A to, with, or against a given amino acid sequence B (which canalternatively be phrased as a given amino acid sequence A that has orcomprises a certain % positives to, with, or against a given amino acidsequence B) is calculated as follows:

100 times the fraction X/Y where X is the number of amino acid residuesscoring a positive value as defined above by the sequence alignmentprogram ALIGN-2 in that program's alignment of A and B, and where Y isthe total number of amino acid residues in B. It will be appreciatedthat where the length of amino acid sequence A is not equal to thelength of amino acid sequence B, the % positives of A to B will notequal the % positives of B to A.

“Isolated,” when used to describe the various polypeptides disclosedherein, means polypeptide that has been identified and separated and/orrecovered from a component of its natural environment. Preferably, theisolated polypeptide is free of association with all components withwhich it is naturally associated. Contaminant components of its naturalenvironment are materials that would typically interfere with diagnosticor therapeutic uses for the polypeptide, and may include enzymes,hormones, and other proteinaceous or non-proteinaceous solutes. Inpreferred embodiments, the polypeptide will be purified (1) to a degreesufficient to obtain at least 15 residues of N-terminal or internalamino acid sequence by use of a spinning cup sequenator, or (2) tohomogeneity by SDS-PAGE under non-reducing or reducing conditions usingCoomassie blue or, preferably, silver stain. Isolated polypeptideincludes polypeptide in situ within recombinant cells, since at leastone component of the PRO10282 (Stra6) natural environment will not bepresent. Ordinarily, however, isolated polypeptide will be prepared byat least one purification step.

An “isolated” nucleic acid molecule encoding a PRO10282 (Stra6)polypeptide is a nucleic acid molecule that is identified and separatedfrom at least one contaminant nucleic acid molecule with which it isordinarily associated in the natural source of the PRO10282-encodingnucleic acid. Preferably, the isolated nucleic is free of associationwith all components with which it is naturally associated. An isolatedPRO10282-encoding nucleic acid molecule is other than in the form orsetting in which it is found in nature. Isolated nucleic acid moleculestherefore are distinguished from the PRO10282-encoding nucleic acidmolecule as it exists in natural cells. However, an isolated nucleicacid molecule encoding a PRO10282 (Stra6) polypeptide includesPRO10282-encoding nucleic acid molecules contained in cells thatordinarily express PRO10282 where, for example, the nucleic acidmolecule is in a chromosomal location different from that of naturalcells.

The term “control sequences” refers to DNA sequences necessary for theexpression of an operably linked coding sequence in a particular hostorganism. The control sequences that are suitable for prokaryotes, forexample, include a promoter, optionally an operator sequence, and aribosome binding site. Eukaryotic cells are known to utilize promoters,polyadenylation signals, and enhancers.

Nucleic acid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For example, DNA for apresequence or secretory leader is operably linked to DNA for apolypeptide if it is expressed as a preprotein that participates in thesecretion of the polypeptide; a promoter or enhancer is operably linkedto a coding sequence if it affects the transcription of the sequence; ora ribosome binding site is operably linked to a coding sequence if it ispositioned so as to facilitate translation. Generally, “operably linked”means that the DNA sequences being linked are contiguous, and, in thecase of a secretory leader, contiguous and in reading phase. However,enhancers do not have to be contiguous. Linking is accomplished byligation at convenient restriction sites. If such sites do not exist,the synthetic oligonucleotide adaptors or linkers are used in accordancewith conventional practice.

The term “antibody” is used in the broadest sense and specificallycovers, for example, single anti-PRO10282 monoclonal antibodies(including agonist, antagonist, and neutralizing antibodies),anti-PRO10282 antibody compositions with polyepitopic specificity,single chain anti-PRO10282 antibodies, and fragments of anti-PRO10282antibodies (see below). The term “monoclonal antibody” as used hereinrefers to an antibody obtained from a population of substantiallyhomogeneous antibodies, i.e., the individual antibodies comprising thepopulation are identical except for possible naturally-occurringmutations that may be present in minor amounts.

“Stringency” of hybridization reactions is readily determinable by oneof ordinary skill in the art, and generally is an empirical calculationdependent upon probe length, washing temperature, and saltconcentration. In general, longer probes require higher temperatures forproper annealing, while shorter probes need lower temperatures.Hybridization generally depends on the ability of denatured DNA toreanneal when complementary strands are present in an environment belowtheir melting temperature. The higher the degree of desired homologybetween the probe and hybridizable sequence, the higher the relativetemperature which can be used. As a result, it follows that higherrelative temperatures would tend to make the reaction conditions morestringent, while lower temperatures less so. For additional details andexplanation of stringency of hybridization reactions, see Ausubel etal., Current Protocols in Molecular Biology, Wiley IntersciencePublishers, (1995).

“Stringent conditions” or “high stringency conditions”, as definedherein, may be identified by those that: (1) employ low ionic strengthand high temperature for washing, for example 0.015 M sodiumchloride/0.0015 M sodium citrate/0.1% sodium dodecyl sulfate at 50° C.;(2) employ during hybridization a denaturing agent, such as formamide,for example, 50% (v/v) formamide with 0.1% bovine serum albumin/0.1%Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5with 750 mM sodium chloride, 75 mM sodium citrate at 42° C.; or (3)employ 50% formamide, 5×SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mMsodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5×Denhardt'ssolution, sonicated salmon sperm DNA (50 g/ml), 0.1% SDS, and 10%dextran sulfate at 42° C., with washes at 42° C. in 0.2×SSC (sodiumchloride/sodium citrate) and 50% formamide at 55° C., followed by ahigh-stringency wash consisting of 0.1×SSC containing EDTA at 55° C.

“Moderately stringent conditions” may be identified as described bySambrook et al., Molecular Cloning: A Laboratory Manual, New York: ColdSpring Harbor Press, 1989, and include the use of washing solution andhybridization conditions (e.g., temperature, ionic strength and % SDS)less stringent that those described above. An example of moderatelystringent conditions is overnight incubation at 37° C. in a solutioncomprising: 20% formamide, 5×SSC (150 mM NaCl, 15 mM trisodium citrate),50 mM sodium phosphate (pH 7.6), 5×Denhardt's solution, 10% dextransulfate, and 20 mg/ml denatured sheared salmon sperm DNA, followed bywashing the filters in 1×SSC at about 37-50° C. The skilled artisan willrecognize bow to adjust the temperature, ionic strength, etc. asnecessary to accommodate factors such as probe length and the like.

The term “epitope tagged” when used herein refers to a chimericpolypeptide comprising a PRO10282 polypeptide fused to a “tagpolypeptide”. The tag polypeptide has enough residues to provide anepitope against which an antibody can be made, yet is short enough suchthat it does not interfere with activity of the polypeptide to which itis fused. The tag polypeptide preferably also is fairly unique so thatthe antibody does not substantially cross-react with other epitopes.Suitable tag polypeptides generally have at least six amino acidresidues and usually between about 8 and 50 amino acid residues(preferably, between about 10 and 20 amino acid residues).

The term “antibody” is used in the broadest sense and specificallycovers, for example, single anti-PRO10282 (anti-Stra6) monoclonalantibodies (including agonist, antagonist, and neutralizing antibodies),anti-PRO10282 antibody compositions with polyepitopic specificity,single chain anti-PRO10282 antibodies, and fragments of anti-PRO10282antibodies (see below).

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally-occurring mutations that may be present inminor amounts.

“Antibody fragments” comprise a portion of an intact antibody,preferably the antigen binding or variable region of the intactantibody. Examples of antibody fragments include Fab, Fab′, F(ab′)₂, andFv fragments; diabodies; linear antibodies (Zapata et al., Protein Eng.,8(10):1057-1062 [1995]); single-chain antibody molecules; andmultispecific antibodies formed from antibody fragments.

Papain digestion of antibodies produces two identical antigen-bindingfragments, called “Fab” fragments, each with a single antigen-bindingsite, and a residual “Fc” fragment, whose name reflects its ability tocrystallize readily. Pepsin treatment yields an F(ab′)₂ fragment thathas two antigen-combining sites and is still capable of cross-linkingantigen.

“Fv” is the minimum antibody fragment, which contains a completeantigen-recognition and -binding site. This region consists of a dimerof one heavy- and one light-chain variable domain in tight, non-covalentassociation. It is in this configuration that the three CDRs of eachvariable domain interact to define an antigen-binding site on thesurface of the V_(H)-V_(L) dimer. Collectively, the six CDRs conferantigen-binding specificity to the antibody. However, even a singlevariable domain (or half of an Fv comprising only three CDRs specificfor an antigen) has the ability to recognize and bind antigen, althoughat a lower affinity than the entire binding site.

The Fab fragment also contains the constant domain of the light chainand the first constant domain (CH1) of the heavy chain. Fab fragmentsdiffer from Fab′ fragments by the addition of a few residues at thecarboxy terminus of the heavy chain CH1 domain including one or morecysteines from the antibody hinge region. Fab′-SH is the designationherein for Fab′ in which the cysteine residue(s) of the constant domainsbear a free thiol group. F(ab′)₂ antibody fragments originally wereproduced as pairs of Fab′ fragments which have hinge cysteines betweenthem. Other chemical couplings of antibody fragments are also known.

The “light chains” of antibodies (immunoglobulins) from any vertebratespecies can be assigned to one of two clearly distinct types, calledkappa ( ) and lambda ( ), based on the amino acid sequences of theirconstant domains.

Depending on the amino acid sequence of the constant domain of theirheavy chains, immunoglobulins can be assigned to different classes.There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, andIgM, and several of these may be further divided into subclasses(isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy-chainconstant domains that correspond to the different classes ofimmunoglobulins are called , , , and, respectively. The subunitstructures and three-dimensional configurations of different classes ofimmunoglobulins are well known.

The term “diabodies” refers to small antibody fragments with twoantigen-binding sites, which fragments comprise a heavy-chain variabledomain (V_(H)) connected to a light-chain variable domain (V_(L)) in thesame polypeptide chain (V_(H)-V_(L)). By using a linker that is tooshort to allow pairing between the two domains on the same chain, thedomains are forced to pair with the complementary domains of anotherchain and create two antigen-binding sites. Diabodies are described morefully in, for example, EP 404,097; WO 93/11161; and Hollinger et al.,Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993).

An “isolated” antibody is one which has been identified and separatedand/or recovered from a component of its natural environment.Contaminant components of its natural environment are materials whichwould interfere with diagnostic or therapeutic uses for the antibody,and may include enzymes, hormones, and other proteinaceous ornonproteinaceous solutes. In preferred embodiments, the antibody will bepurified (1) to greater than 95% by weight of antibody as determined bythe Lowry method, and most preferably more than 99% by weight, (2) to adegree sufficient to obtain at least 15 residues of N-terminal orinternal amino acid sequence by use of a spinning cup sequenator, or (3)to homogeneity by SDS-PAGE under reducing or nonreducing conditionsusing Coomassie blue or, preferably, silver stain. Isolated antibodyincludes the antibody in situ within recombinant cells since at leastone component of the antibody's natural environment will not be present.Ordinarily, however, isolated antibody will be prepared by at least onepurification step.

The term “variable” refers to the fact that certain portions of thevariable domains differ extensively in sequence among antibodies and areused in the binding and specificity of each particular antibody for itsparticular antigen. However, the variability is not evenly distributedthroughout the variable domains of antibodies. It is concentrated inthree segments called complementarity-determining regions (CDRs) orhypervariable regions both in the light-chain and the heavy-chainvariable domains. The more highly conserved portions of variable domainsare called the framework (FR) regions. The variable domains of nativeheavy and light chains each comprise four FR regions, largely adopting a-sheet configuration, connected by three CDRs, which form loopsconnecting, and in some cases forming part of, the -sheet structure. TheCDRs in each chain are held together in close proximity by the FRregions and, with the CDRs from the other chain, contribute to theformation of the antigen-binding site of antibodies (see Kabat et al.,NIH Publ. No. 91-3242, Vol. 1, pages 647-669 (1991)). The constantdomains are not involved directly in binding an antibody to an antigen,but exhibit various effector functions, such as participation of theantibody in antibody-dependent cellular toxicity.

The term “hypervariable region” when used herein refers to the aminoacid residues of an antibody which are responsible for antigen-binding.The hypervariable region comprises amino acid residues from a“complementarity determining region” or “CDR” (i.e., residues 24-34(L1), 50-56 (L2) and 89-97 (L3) in the light chain variable domain and31-35 (H1), 50-65 (H2) and 95-102 (H3) in the heavy chain variabledomain; Kabat et al., Sequences of Proteins of Immunological Interest,5th Ed. Public Health Service, National Institute of Health, Bethesda,Md. [1991]) and/or those residues from a “hypervariable loop” (i.e.,residues 26-32 (L1), 50-52 (L2) and 91-96 (L3) in the light chainvariable domain and 26-32 (H1), 53-55 (H2) and 96-101 (H3) in the heavychain variable domain; Clothia and Lesk, J. Mol. Biol., 196:901-917[1987]). “Framework” or “FR” residues are those variable domain residuesother than the hypervariable region residues as herein defined.

“Humanized” forms of non-human (e.g., murine) antibodies are chimericimmunoglobulins, immunoglobulin chains or fragments thereof (such as Fv,Fab, Fab′, F(ab′)₂ or other antigen-binding subsequences of antibodies)which contain minimal sequence derived from non-human immunoglobulin.For the most part, humanized antibodies are human immunoglobulins(recipient antibody) in which residues from a CDR of the recipient arereplaced by residues from a CDR of a non-human species (donor antibody)such as mouse, rat or rabbit having the desired specificity, affinity,and capacity. In some instances, Fv FR residues of the humanimmunoglobulin are replaced by corresponding non-human residues.Furthermore, humanized antibodies may comprise residues which are foundneither in the recipient antibody nor in the imported CDR or frameworksequences. These modifications are made to further refine and maximizeantibody performance. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the CDR regions correspond to thoseof a non-human immunoglobulin and all or substantially all of the FRregions are those of a human immunoglobulin sequence. The humanizedantibody optimally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin. For further details, see, Jones et. al., Nature,321:522-525 (1986); Reichmann et al., Nature, 332:323-329 [1988]; andPresta, Curr. Op. Struct. Biol., 2:593-596 (1992). The humanizedantibody includes a PRIMATIZED™ antibody wherein the antigen-bindingregion of the antibody is derived from an antibody produced byimmunizing macaque monkeys with the antigen of interest.

As used herein, the term “immunoadhesin” designates antibody-likemolecules which combine the binding specificity of a heterologousprotein (an “adhesin”) with the effector functions of immunoglobulinconstant domains. Structurally, the immunoadhesins comprise a fusion ofan amino acid sequence with the desired binding specificity which isother than the antigen recognition and binding site of an antibody(i.e., is “heterologous”), and an immunoglobulin constant domainsequence. The adhesin part of an immunoadhesin molecule typically is acontiguous amino acid sequence comprising at least the binding site of areceptor or a ligand. The immunoglobulin constant domain sequence in theimmunoadhesin may be obtained from any immunoglobulin, such as IgG-1,IgG-2, IgG-3, or IgG-4 subtypes, IgA (including IgA-1 and IgA-2), IgE,IgD or IgM.

“Active” or “activity” for the purposes herein refers to form(s) ofPRO10282 which retain a biological and/or an immunological activity ofnative or naturally-occurring PRO10282, wherein “biological” activityrefers to a biological function (either inhibitory or stimulatory)caused by a native or naturally-occurring PRO10282 other than theability to induce the production of an antibody against an antigenicepitope possessed by a native or naturally-occurring PRO10282 and an“immunological” activity refers to the ability to induce the productionof an antibody against an antigenic epitope possessed by a native ornaturally-occurring PRO10282. A preferred biological activity includes,for example, a role in growth, development or differentiation inresponse to retinoic acid. Based on close similarity with Stra6, amurine protein induced in response to retinoic acid, PRO10282polypeptide disclosed herein may play an important role, for example, inearly dorsoventral limb patterning and later in the control ofendochondral ossification. Another biological activity is involvement intumor development and growth.

“Immunological activity” preferably is “immunological cross-reactivity,”which is used to mean that the candidate polypeptide is capable ofcompetitively inhibiting the qualitative biological activity of aPRO10282 (Stra6) polypeptide having this activity with polyclonalantisera raised against the known active PRO10282 (Stra6) polypeptide.Such antisera are prepared in conventional fashion by injecting goats orrabbits, for example, subcutaneously with the known active analogue incomplete Freund's adjuvant, followed by booster intraperitoneal orsubcutaneous injection in incomplete Freund's. The immunologicalcross-reactivity is preferably “specific”, which means that the bindingaffinity of the immunologically cross-reactive molecule (e.g. antibody)identified, to the corresponding Stra6 polypeptide is significantlyhigher (preferably at least about 2-times, more preferably at leastabout 4-times, even more preferably at least about 8-times, mostpreferably at least about 10-times higher) than the binding affinity ofthat molecule to any other known native polypeptide.

The term “antagonist” is used in the broadest sense, and includes anymolecule that partially or fully blocks, inhibits, or neutralizes abiological activity of a native PRO10282 polypeptide disclosed herein.In a similar manner, the term “agonist” is used in the broadest senseand includes any molecule that mimics a biological activity of a nativePRO10282 polypeptide disclosed herein. Suitable agonist or antagonistmolecules specifically include agonist or antagonist antibodies orantibody fragments, fragments or amino acid sequence variants of nativePRO10282 polypeptides, peptides, small organic molecules, etc. Methodsfor identifying agonists or antagonists of a PRO10282 polypeptide maycomprise contacting a PRO10282 polypeptide with a candidate agonist orantagonist molecule and measuring a detectable change in one or morebiological activities normally associated with the PRO10282 polypeptide.

“Tumor”, as used herein, refers to all neoplastic cell growth andproliferation, whether malignant or benign, and all pre-cancerous andcancerous cells and tissues.

The terms “cancer” and “cancerous” refer to or describe thephysiological condition in mammals that is typically characterized byunregulated cell growth. Examples of cancer include but are not limitedto, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. Moreparticular examples of such cancers include breast cancer, prostatecancer, colon cancer, squamous cell cancer, small-cell lung cancer,non-small cell lung cancer, gastrointestinal cancer, pancreatic cancer,glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladdercancer, hepatoma, colorectal cancer, endometrial carcinoma, salivarygland carcinoma, kidney cancer, liver cancer, vulval cancer, thyroidcancer, hepatic carcinoma and various types of head and neck cancer.

“Treatment” refers to both therapeutic treatment and prophylactic orpreventative measures, wherein the object is to prevent or slow down(lessen) the targeted pathologic condition or disorder. Those in need oftreatment include those already with the disorder as well as those proneto have the disorder or those in whom the disorder is to be prevented.In tumor (e.g., cancer) treatment, a therapeutic agent may directlydecrease the pathology of tumor cells, or render the tumor cells moresusceptible to treatment by other therapeutic agents, e.g., radiationand/or chemotherapy.

The “pathology” of cancer includes all phenomena that compromise thewell-being of the patient. This includes, without limitation, abnormalor uncontrollable cell growth, metastasis, interference with the normalfunctioning of neighboring cells, release of cytokines or othersecretory products at abnormal levels, suppression or aggravation ofinflammatory or immunological response, etc.

“Chronic” administration refers to administration of the agent(s) in acontinuous mode as opposed to an acute mode, so as to maintain theinitial therapeutic effect (activity) for an extended period of time.“Intermittent” administration is treatment that is not consecutivelydone without interruption, but rather is cyclic in nature.

“Mammal” for purposes of treatment refers to any animal classified as amammal, including humans, domestic and farm animals, and zoo, sports, orpet animals, such as dogs, cats, cattle, horses, sheep, pigs, goats,rabbits, etc. Preferably, the mammal is human.

Administration “in combination with” one or more further therapeuticagents includes simultaneous (concurrent) and consecutive administrationin any order.

“Carriers” as used herein include pharmaceutically acceptable carriers,excipients, or stabilizers which are nontoxic to the cell or mammalbeing exposed thereto at the dosages and concentrations employed. Oftenthe physiologically acceptable carrier is an aqueous pH bufferedsolution. Examples of physiologically acceptable carriers includebuffers such as phosphate, citrate, and other organic acids;antioxidants including ascorbic acid; low molecular weight (less thanabout 10 residues) polypeptide; proteins, such as serum albumin,gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, arginine or lysine; monosaccharides, disaccharides, andother carbohydrates including glucose, mannose, or dextrins; chelatingagents such as EDTA; sugar alcohols such as mannitol or sorbitol;salt-forming counterions such as sodium; and/or nonionic surfactantssuch as TWEEN™, polyethylene glycol (PEG), and PLURONICS™.

The word “label” when used herein refers to a detectable compound orcomposition which is conjugated directly or indirectly to the antibodyso as to generate a “labeled” antibody. The label may be detectable byitself (e.g. radioisotope labels or fluorescent labels) or, in the caseof an enzymatic label, may catalyze chemical alteration of a substratecompound or composition which is detectable.

By “solid phase” is meant a non-aqueous matrix to which the antibody ofthe present invention can adhere. Examples of solid phases encompassedherein include those formed partially or entirely of glass (e.g.,controlled pore glass), polysaccharides (e.g., agarose),polyacrylamides, polystyrene, polyvinyl alcohol and silicones. Incertain embodiments, depending on the context, the solid phase cancomprise the well of an assay plate; in others it is a purificationcolumn (e.g., an affinity chromatography column). This term alsoincludes a discontinuous solid phase of discrete particles, such asthose described in U.S. Pat. No. 4,275,149.

A “liposome” is a small vesicle composed of various types of lipids,phospholipids and/or surfactant which is useful for delivery of a drug(such as a PRO10282 polypeptide or antibody thereto) to a mammal. Thecomponents of the liposome are commonly arranged in a bilayer formation,similar to the lipid arrangement of biological membranes.

A “small molecule” is defined herein to have a molecular weight belowabout 500 Daltons.

The phrases “gene amplification” and “gene duplication” are usedinterchangeably and refer to a process by which multiple copies of agene or gene fragment are formed in a particular cell or cell line. Theduplicated region (a stretch of amplified DNA) is often referred to as“amplicon.” Usually, the amount of the messenger RNA (mRNA) produced,i.e., the level of gene expression, also increases in the proportion ofthe number of copies made of the particular gene expressed.

The term “cytotoxic agent” as used herein refers to a substance thatinhibits or prevents the function of cells and/or causes destruction ofcells. The term is intended to include radioactive isotopes (e.g., I¹³¹,I¹²⁵, Y⁹⁰ and Re¹⁸⁶), chemotherapeutic agents, and toxins such asenzymatically active toxins of bacterial, fungal, plant or animalorigin, or fragments thereof.

A “chemotherapeutic agent” is a chemical compound useful in thetreatment of cancer. Examples of chemotherapeutic agents includeadriamycin, doxorubicin, epirubicin, 5-fluorouracil, cytosinearabinoside (“Ara-C”), cyclophosphamide, thiotepa, busulfan, cytoxin,taxoids, e.g., paclitaxel (Taxol, Bristol-Myers Squibb Oncology,Princeton, N.J.), and doxetaxel (Taxotere, Rhône-Poulenc Rorer, Antony,Rnace), toxotere, methotrexate, cisplatin, melphalan, vinblastine,bleomycin, etoposide, ifosfamide, mitomycin C, mitoxantrone,vincristine, vinorelbine, carboplatin, teniposide, daunomycin,caminomycin, aminopterin, dactinomycin, mitomycins, esperamicins (seeU.S. Pat. No. 4,675,187), 5-FU, 6-thioguanine, 6-mercaptopurine,actinomycin D, VP-16, chlorambucil, melphalan, and other relatednitrogen mustards. Also included in this definition are hormonal agentsthat act to regulate or inhibit hormone action on tumors such astamoxifen and onapristone.

A “growth inhibitory agent” when used herein refers to a compound orcomposition which inhibits growth of a cell, especially cancer celloverexpressing any of the genes identified herein, either in vitro or invivo. Thus, the growth inhibitory agent is one which significantlyreduces the percentage of cells overexpressing such genes in S phase.Examples of growth inhibitory agents include agents that block cellcycle progression (at a place other than S phase), such as agents thatinduce G1 arrest and M-phase arrest. Classical M-phase blockers includethe vincas (vincristine and vinblastine), taxol, and topo II inhibitorssuch as doxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin.Those agents that arrest G1 also spill over into S-phase arrest, forexample, DNA alkylating agents such as tamoxifen, prednisone,dacarbazine, mechlorethamine, cisplatin, methotrexate, 5-fluorouracil,and ara-C. Further information can be found in The Molecular Basis ofCancer, Mendelsohn and Israel, eds., Chapter 1, entitled “Cell cycleregulation, oncogens, and antineoplastic drugs” by Murakami et al., (WBSaunders: Philadelphia, 1995), especially p. 13.

“Doxorubicin” is an anthracycline antibiotic. The full chemical name ofdoxorubicin is(8S-cis)-10-[(3-amino-2,3,6-trideoxy-1-L-lyxo-hexapyranosyl)oxy]-7,8,9,10-tetrahydro-6,8,11-trihydroxy-8-(hydroxyacetyl)-1-methoxy-5,12-naphthacenedione.

The term “cytokine” is a generic term for proteins released by one cellpopulation which act on another cell as intercellular mediators.Examples of such cytokines are lymphokines, monokines, and traditionalpolypeptide hormones. Included among the cytokines are growth hormonesuch as human growth hormone, N-methionyl human growth hormone, andbovine growth hormone; parathyroid hormone; thyroxine; insulin;proinsulin; relaxin; prorelaxin; glycoprotein hormones such as folliclestimulating hormone (FSH), thyroid stimulating hormone (TSH), andluteinizing hormone (LH); hepatic growth factor; fibroblast growthfactor; prolactin; placental lactogen; tumor necrosis factor- and-;mullerian-inhibiting substance; mouse gonadotropin-associated peptide;inhibin; activin; vascular endothelial growth factor; integrin;thrombopoietin (TPO); nerve growth factors such as NGF-; platelet-growthfactor; transforming growth factors (TGFs) such as TGF- and TGF-;insulin-like growth factor-I and -II; erythropoietin (EPO);osteoinductive factors; interferons such as interferon-, -, and-; colonystimulating factors (CSFs) such as macrophage-CSF (M-CSF);granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF);interleukins (ILs) such as IL-1, IL-1a, IL-2, IL-3, IL-4, IL-5, IL-6,IL-7, IL-8, IL-9, IL-11, IL-12; a tumor necrosis factor such as TNF- orTNF-B; and other polypeptide factors including LIF and kit ligand (KL).As used herein, the term cytokine includes proteins from natural sourcesor from recombinant cell culture and biologically active equivalents ofthe native sequence cytokines.

The term “prodrug” as used in this application refers to a precursor orderivative form of a pharmaceutically active substance that is lesscytotoxic to tumor cells compared to the parent drug and is capable ofbeing enzymatically activated or converted into the more active parentform. See, e.g., Wilman, “Prodrugs in Cancer Chemotherapy”, BiochemicalSociety Transactions, 14:375-382, 615th Meeting, Belfast (1986), andStella et al., “Prodrugs: A Chemical Approach to Targeted DrugDelivery”, Directed Drug Delivery, Borchardt et al., (ed.), pp. 147-267,Humana Press (1985). The prodrugs of this invention include, but are notlimited to, phosphate-containing prodrugs, thiophosphate-containingprodrugs, sulfate-containing prodrugs, peptide-containing prodrugs,D-amino acid-modified prodrugs, glysocylated prodrugs,B-lactam-containing prodrugs, optionally substitutedphenoxyacetamide-containing prodrugs or optionally substitutedphenylacetamide-containing prodrugs, 5-fluorocytosine and other5-fluorouridine prodrugs which can be converted into the more activecytotoxic free drug. Examples of cytotoxic drugs that can be derivatizedinto a prodrugs form for use in this invention include, but are notlimited to, those chemotherapeutic agents described above.

An “effective amount” of a polypeptide disclosed herein or an antagonistthereof, in reference to inhibition of neoplastic cell growth, tumorgrowth or cancer cell growth, is an amount capable of inhibiting, tosome extent, the growth of target cells. The term includes an amountcapable of invoking a growth inhibitory, cytostatic and/or cytotoxiceffect and/or apoptosis of the target cells. An “effective amount” of aPRO10282 (including PRO19578) polypeptide or an antagonist thereof forthe purposes of inhibiting neoplastic cell growth, tumor cell or cancercell growth, may be determined empirically and in a routine manner.

A “therapeutically effective amount”, in reference to the treatment oftumor, refers to an amount capable of invoking one or more of thefollowing effects: (1) inhibition, to some extent, of tumor growth,including, slowing down and complete growth arrest; (2) reduction in thenumber of tumor cells; (3) reduction in tumor size; (4) inhibition(i.e., reduction, slowing down or complete stopping) of tumor cellinfiltration into peripheral organs; (5) inhibition (i.e., reduction,slowing down or complete stopping) of metastasis; (6) enhancement ofanti-tumor immune response, which may, but does not have to, result inthe regression or rejection of the tumor; and/or (7) relief, to someextent, of one or more symptoms associated with the disorder. A“therapeutically effective amount” of a PRO10282 polypeptide antagonistfor purpose of treatment of tumor may be determined empirically and in aroutine manner.

A “growth inhibitory amount” of a PRO10282 antagonist is an amountcapable of inhibiting the growth of a cell, especially tumor, e.g.cancer cell, either in vitro or in vivo.

A “cytotoxic amount” of a PRO10282 antagonist for purposes of inhibitingneoplastic cell growth may be determined empirically and in a routinemanner.

A “cytotoxic amount” or a PRO10282 antagonist is an amount capable ofcausing the destruction of a cell, especially tumor, e.g. cancer cell,either in vitro or in vivo. A “cytotoxic amount” of a PRO10282antagonist for purposes of inhibiting neoplastic cell growth may bedetermined empirically and in a routine manner.

“Antisense oligodeoxynucleotides” or “antisense oligonucleotides” (whichterms are used interchangeably) are defined as nucleic acid moleculesthat can inhibit the transcription and/or translation of target genes ina sequence-specific manner. The term “antisense” refers to the fact thatthe nucleic acid is complementary t the coding (“sense”) geneticsequence of the target gene. Antisense oligonucleotides hybridize in anantiparallel orientation to nascent mRNA through Watson-Crickbase-pairing. By binding the target mRNA template, antisenseoligonucleotides block the successful translation of the encodedprotein. The term specifically includes antisense agents called“ribozomes” that have been designated to induce catalytic cleavage of atarget RNA by addition of a sequence that has natural self-splicingactivity (Warzocha and Wotowiec, “Antisense strategy” biological utilityand prospects in the treatment of hematological malignancies.” Leuk.Lymphoma 24:267-281 [1997]).

II. Compositions and Methods of the Invention

A. Full-Length Native Sequence Human PRO10282 and PRO19578 (Stra6)Polypeptides

The present invention provides newly identified and isolated nucleotidesequences encoding polypeptides referred to in the present applicationas native sequence human PRO10282 (or also UNQ3126) and PRO19578,respectively. In particular, cDNA encoding the native human PRO10282 andPRO19578 polypeptides has been identified and isolated, as disclosed infurther detail in the Examples below. It is noted that proteins producedin separate expression rounds may be given different PRO numbers but theUNQ number is unique for any given DNA and the encoded protein, and willnot be changed. However, for sake of simplicity, in the presentspecification the protein encoded by DNA148380-2827 as well as allfurther native homologues and variants included in the foregoingdefinition of PRO10282, will be referred to as “PRO10282”, regardless oftheir origin or mode of preparation. The polypeptide encoded byDNA148389-2827-1, which is a native variant of “PRO10282,” will bespecifically referred to as “PRO19578.”

As disclosed in the Examples below, cDNA clones designated herein asDNA148380-2827 and DNA148389-2827-1 have been deposited with the ATCC.The actual nucleotide sequence of the clone can readily be determined bythe skilled artisan by sequencing of the deposited clone using routinemethods in the art. The predicted amino acid sequence can be determinedfrom the nucleotide sequence using routine skill. For the full-lengthnative human PRO10282 and PRO19578 polypeptides and encoding nucleicacid described herein, Applicants have identified what is believed to bethe reading frame best identifiable with the sequence informationavailable at the time.

Using the ALIGN-2 sequence alignment computer program referenced above,it has been found that the full-length native sequence PRO10282 (shownin FIG. 2 and SEQ ID NO:2) has certain amino acid sequence identity withStra6, a murine protein induced in response to retinoic acid (AF062476).Accordingly, it is presently believed that the PRO10282 polypeptidedisclosed in the present application is a newly identified member of theStra6 protein family and may possess one or more biological and/orimmunological activities or properties typical of that protein family.PRO19578 is currently believed to be an alternatively spliced variant ofnative, full-length human PRO10282, which has 9 amino acids (SPVDFLAGD)deleted at positions 89-97 of the native human PRO10282 amino acidsequence, and contains Ile (I) instead of Met (M) at position 518, whichcorresponds to position 527 of PRO10282. This indicates that Stra6 maybe polymorphic.

B. PRO10282 (Stra6) Variants

Two specific native sequence human Stra6 polypeptides (native sequencehuman PRO10282 and PRO19578) are disclosed herein. As noted above,PRO19578 is believed to be an alternatively spliced nativa variant ofnative sequence human PRO10282 and is, therefore, a “PRO10282 variant.”In addition to the full-length native sequence PRO10282 and PRO19578polypeptides described herein, it is contemplated that PRO10282 andPRO19578 variants can be prepared. PRO10282 and PRO19578 variants can beprepared by introducing appropriate nucleotide changes into the PRO10282or PRO19578 DNA, and/or by synthesis of the desired PRO10282 variantpolypeptide. Those skilled in the art will appreciate that amino acidchanges may alter post-translational processes of the PRO10282 orPRO19578, such as changing the number or position of glycosylation sitesor altering the membrane anchoring characteristics.

Variations in the native full-length sequence PRO10282, PRO19578 or invarious domains of the PRO10282 or PRO19578 described herein, can bemade, for example, using any of the techniques and guidelines forconservative and non-conservative mutations set forth, for instance, inU.S. Pat. No. 5,364,934. Variations may be a substitution, deletion orinsertion of one or more codons encoding the PRO10282 or PRO19578 thatresults in a change in the amino acid sequence of the PRO10282 orPRO19578 as compared with the native sequence PRO10282 or PRO19578.Optionally the variation is by substitution of at least one amino acidwith any other amino acid in one or more of the domains of the PRO10282or PRO19578. Guidance in determining which amino acid residue may beinserted, substituted or deleted without adversely affecting the desiredactivity may be found by comparing the sequence of the PRO10282 orPRO19578 with that of homologous known protein molecules and minimizingthe number of amino acid sequence changes made in regions of highhomology. Amino acid substitutions can be the result of replacing oneamino acid with another amino acid having similar structural and/orchemical properties, such as the replacement of a leucine with a serine,i.e., conservative amino acid replacements. Insertions or deletions mayoptionally be in the range of about 1 to 5 amino acids. The variationallowed may be determined by systematically making insertions, deletionsor substitutions of amino acids in the sequence and testing theresulting variants for activity exhibited by the full-length or maturenative sequence.

PRO10282 polypeptide fragments are provided herein. Such fragments maybe truncated at the N-terminus or C-terminus, or may lack internalresidues, for example, when compared with a full-length native protein.Certain fragments lack amino acid residues that are not essential for adesired biological activity of the PRO10282 or PRO19578 polypeptide.

PRO10282 or PRO19578 fragments may be prepared by any of a number ofconventional techniques. Desired peptide fragments may be chemicallysynthesized. An alternative approach involves generating PRO10282 orPRO19578 fragments by enzymatic digestion, e.g., by treating the proteinwith an enzyme known to cleave proteins at sites defined by particularamino acid residues, or by digesting the DNA with suitable restrictionenzymes and isolating the desired fragment. Yet another suitabletechnique involves isolating and amplifying a DNA fragment encoding adesired polypeptide fragment, by polymerase chain reaction (PCR).Oligonucleotides that define the desired termini of the DNA fragment areemployed at the 5′ and 3′ primers in the PCR. Preferably, PRO10282 orPRO19578 polypeptide fragments share at least one biological and/orimmunological activity with the native PRO10282 or PRO19578 polypeptideshown in FIG. 2 (SEQ ID NO:2).

In particular embodiments, conservative substitutions of interest areshown in Table 1 under the heading of preferred substitutions. If suchsubstitutions result in a change in biological activity, then moresubstantial changes, denominated exemplary substitutions in Table 1, oras further described below in reference to amino acid classes, areintroduced and the products screened.

TABLE 1 Original Exemplary Preferred Residue Substitutions SubstitutionsAla (A) val; leu; ile val Arg (R) lys; gln; asn lys Asn (N) gln; his;lys; arg gln Asp (D) glu glu Cys (C) ser ser Gln (Q) asn asn Glu (E) aspasp Gly (G) pro; ala ala His (H) asn; gln; lys; arg arg Ile (I) leu;val; met; ala; phe; leu norleucine Leu (L) norleucine; ile; val; ilemet; ala; phe Lys (K) arg; gln; asn arg Met (M) leu; phe; ile leu Phe(F) leu; val; ile; ala; tyr leu Pro (P) ala ala Ser (S) thr thr Thr (T)ser ser Trp (W) tyr; phe tyr Tyr (Y) trp; phe; thr; ser phe Val (V) ile;leu; met; phe; leu ala; norleucine

Substantial modifications in function or immunological identity of thePRO10282 polypeptide are accomplished by selecting substitutions thatdiffer significantly in their effect on maintaining (a) the structure ofthe polypeptide backbone in the area of the substitution, for example,as a sheet or helical conformation, (b) the charge or hydrophobicity ofthe molecule at the target site, or (c) the bulk of the side chain.Naturally occurring residues are divided into groups based on commonside-chain properties:

(1) hydrophobic: norleucine, met, ala, val, leu, ile;(2) neutral hydrophilic: cys, ser, thr;(3) acidic: asp, glu;(4) basic: asn, gin, his, lys, arg;(5) residues that influence chain orientation: gly, pro; and(6) aromatic: trp, tyr, phe.

Non-conservative substitutions will entail exchanging a member of one ofthese classes for another class. Such substituted residues also may beintroduced into the conservative substitution sites or, more preferably,into the remaining (non-conserved) sites.

The variations can be made using methods known in the art such asoligonucleotide-mediated (site-directed) mutagenesis, alanine scanning,and PCR mutagenesis. Site-directed mutagenesis [Carter et al., Nucl.Acids Res., 13:4331 (1986); Zoller et al., Nucl. Acids Res., 10:6487(1987)], cassette mutagenesis [Wells et al., Gene, 34:315 (1985)],restriction selection mutagenesis [Wells et al., Philos. Trans. R. Soc.London SerA, 317:415 (1986)] or other known techniques can be performedon the cloned DNA to produce the PRO10282 variant DNA.

Scanning amino acid analysis can also be employed to identify one ormore amino acids along a contiguous sequence. Among the preferredscanning amino acids are relatively small, neutral amino acids. Suchamino acids include alanine, glycine, serine, and cysteine. Alanine istypically a preferred scanning amino acid among this group because iteliminates the side-chain beyond the beta-carbon and is less likely toalter the main-chain conformation of the variant [Cunningham and Wells,Science, 244: 1081-1085 (1989)]. Alanine is also typically preferredbecause it is the most common amino acid. Further, it is frequentlyfound in both buried and exposed positions [Creighton, The Proteins,(W.H. Freeman & Co., N.Y.); Chothia, J. Mol. Biol., 150:1 (1976)]. Ifalanine substitution does not yield adequate amounts of variant, anisoteric amino acid can be used.

C. Modifications of Native and Variant PRO10282 (Stra6)

Covalent modifications of PRO10282 and PRO10282 variants, includingPRO19578, are included within the scope of this invention. One type ofcovalent modification includes reacting targeted amino acid residues ofa PRO10282 polypeptide with an organic derivatizing agent that iscapable of reacting with selected side chains or the N- or C-terminalresidues of the PRO10282. Derivatization with bifunctional agents isuseful, for instance, for crosslinking PRO10282 to a water-insolublesupport matrix or surface for use in the method for purifyinganti-PRO10282 antibodies, and vice-versa. Commonly used crosslinkingagents include, e.g., 1,1-bis(diazoacetyl)-2-phenylethane,glutaraldehyde, N-hydroxysuccinimide esters, for example, esters with4-azidosalicylic acid, homobifunctional imidoesters, includingdisuccinimidyl esters such as 3,3′-dithiobis(succinimidylpropionate),bifunctional maleimides such as bis-N-maleimido-1,8-octane and agentssuch as methyl-3-[(p-azidophenyl)dithio]propioimidate.

Other modifications include deamidation of glutaminyl and asparaginylresidues to the corresponding glutamyl and aspartyl residues,respectively, hydroxylation of proline and lysine, phosphorylation ofhydroxyl groups of seryl or threonyl residues, methylation of the -aminogroups of lysine, arginine, and histidine side chains [T. E. Creighton,Proteins: Structure and Molecular Properties, W.H. Freeman & Co., SanFrancisco, pp. 79-86 (1983)], acetylation of the N-terminal amine, andamidation of any C-terminal carboxyl group.

Another type of covalent modification of the PRO10282 polypeptidesincluded within the scope of this invention comprises altering thenative glycosylation pattern of the polypeptide. Native sequencePRO10282 and PRO19578 polypeptides contain an N-linked glycosylationsite at positions 8-12 of their amino acid sequence. “Altering thenative glycosylation pattern” is intended for purposes herein to meandeleting one or more carbohydrate moieties found in a native sequencePRO10282 or PRO19578 (either by removing the underlying glycosylationsite or by deleting the glycosylation by chemical and/or enzymaticmeans), and/or adding one or more glycosylation sites that are notpresent in the native sequence PRO10282 or PRO19578. In addition, thephrase includes qualitative changes in the glycosylation of the nativeproteins, involving a change in the nature and proportions of thevarious carbohydrate moieties present.

Addition of glycosylation sites to a native or variant PRO10282polypeptide may be accomplished by altering the amino acid sequence. Thealteration may be made, for example, by the addition of, or substitutionby, one or more serine or threonine residues to the native sequencePRO10282 (for O-linked glycosylation sites) The PRO10282 amino acidsequence may optionally be altered through changes at the DNA level,particularly by mutating the DNA encoding the PRO10282 polypeptide atpreselected bases such that codons are generated that will translateinto the desired amino acids.

Another means of increasing the number of carbohydrate moieties on thePRO10282 polypeptide is by chemical or enzymatic coupling of glycosidesto the polypeptide. Such methods are described in the art, e.g., in WO87/05330 published 11 Sep. 1987, and in Aplin and Wriston, CRC Crit.Rev. Biochem., pp. 259-306 (1981).

Removal of carbohydrate moieties present on the PRO10282 polypeptide maybe accomplished chemically or enzymatically or by mutationalsubstitution of codons encoding for amino acid residues that serve astargets for glycosylation. Chemical deglycosylation techniques are knownin the art and described, for instance, by Hakimuddin, et al., Arch.Biochem. Biophys., 259:52 (1987) and by Edge et al., Anal. Biochem.,118:131 (1981). Enzymatic cleavage of carbohydrate moieties onpolypeptides can be achieved by the use of a variety of endo- andexo-glycosidases as described by Thotakura et al., Meth. Enzymol.,138:350 (1987).

Another type of covalent modification of PRO10282 comprises linking thePRO10282 polypeptide to one of a variety of nonproteinaceous polymers,e.g., polyethylene glycol (PEG), polypropylene glycol, orpolyoxyalkylenes, in the manner set forth in U.S. Pat. No. 4,640,835;4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337.

The PRO10282 polypeptides of the present invention may also be modifiedin a way to form a chimeric molecule comprising a PRO10282 fused toanother, heterologous polypeptide or amino acid sequence.

In one embodiment, such a chimeric molecule comprises a fusion of thePRO10282 with a tag polypeptide which provides an epitope to which ananti-tag antibody can selectively bind. The epitope tag is generallyplaced at the amino- or carboxyl-terminus of the PRO10282. The presenceof such epitope-tagged forms of the PRO10282 can be detected using anantibody against the tag polypeptide. Also, provision of the epitope tagenables the PRO10282 to be readily purified by affinity purificationusing an anti-tag antibody or another type of affinity matrix that bindsto the epitope tag. Various tag polypeptides and their respectiveantibodies are well known in the art. Examples include poly-histidine(poly-his) or poly-histidine-glycine (poly-his-gly) tags; the flu HA tagpolypeptide and its antibody 12CA5 [Field et al., Mol. Cell. Biol.,8:2159-2165 (1988)]; the c-myc tag and the 8F9, 3C7, 6E10, G4, B7 and9E10 antibodies thereto [Evan et al., Molecular and Cellular Biology,5:3610-3616 (1985)]; and the Herpes Simplex virus glycoprotein D (gD)tag and its antibody [Paborsky et al., Protein Engineering, 3(6):547-553(1990)]. Other tag polypeptides include the Flag-peptide [Hopp et al.,BioTechnology, 6:1204-1210 (1988)]; the KT3 epitope peptide [Martin etal., Science, 255:192-194 (1992)]; an -tubulin epitope peptide [Skinneret al., J. Biol. Chem., 266: 15163-15166 (1991)]; and the T7 gene 10protein peptide tag [Lutz-Freyermuth et al., Proc. Natl. Acad. Sci. USA,87:6393-6397 (1990)].

In an alternative embodiment, the chimeric molecule may comprise afusion of the PRO10282 with an immunoglobulin or a particular region ofan immunoglobulin. For a bivalent form of the chimeric molecule (alsoreferred to as an “immunoadhesin”), such a fusion could be to the Fcregion of an IgG molecule. The Ig fusions preferably include thesubstitution of a soluble (transmembrane domain deleted or inactivated)form of a PRO10282 polypeptide in place of at least one variable regionwithin an Ig molecule. In a particularly preferred embodiment, theimmunoglobulin fusion includes the hinge, CH2 and CH3, or the hinge,CH1, CH2 and CH3 regions of an IgG1 molecule. For the production ofimmunoglobulin fusions see also U.S. Pat. No. 5,428,130 issued Jun. 27,1995.

D. Preparation of Naitge or Variant PRO10282 (Stra6) Polypeptide

The description below relates primarily to production of native sequenceand variant PRO10282 (Stra6) polypeptides, specifically includingPRO19578, by culturing cells transformed or transfected with a vectorcontaining PRO10282 nucleic acid. It is, of course, contemplated thatalternative methods, which are well known in the art, may be employed toprepare PRO10282. For instance, the PRO10282 sequence, or portionsthereof, may be produced by direct peptide synthesis using solid-phasetechniques [see, e.g., Stewart et al., Solid-Phase Peptide Synthesis,W.H. Freeman Co., San Francisco, Calif. (1969); Merrifield, J. Am. Chem.Soc., 85:2149-2154 (1963)]. In vitro protein synthesis may be performedusing manual techniques or by automation. Automated synthesis may beaccomplished, for instance, using an Applied Biosystems PeptideSynthesizer (Foster City, Calif.) using manufacturer's instructions.Various portions of the PRO10282 may be chemically synthesizedseparately and combined using chemical or enzymatic methods to producethe full-length PRO10282.

1. Isolation of DNA Encoding PRO10282 (Stra6)

DNA encoding PRO10282 may be obtained from a cDNA library prepared fromtissue believed to possess the PRO10282 mRNA and to express it at adetectable level. Accordingly, human PRO10282 DNA can be convenientlyobtained from a cDNA library prepared from human tissue, such asdescribed in the Examples. The PRO10282-encoding gene may also beobtained from a genomic library or by known synthetic procedures (e.g.,automated nucleic acid synthesis).

Libraries can be screened with probes (such as antibodies to thePRO10282 or oligonucleotides of at least about 20-80 bases) designed toidentify the gene of interest or the protein encoded by it. Screeningthe cDNA or genomic library with the selected probe may be conductedusing standard procedures, such as described in Sambrook et al.,Molecular Cloning: A Laboratory Manual (New York: Cold Spring HarborLaboratory Press, 1989). An alternative means to isolate the geneencoding PRO10282 is to use PCR methodology [Sambrook et al., supra;Dieffenbach et al., PCR Primer: A Laboratory Manual (Cold Spring HarborLaboratory Press, 1995)].

The Examples below describe techniques for screening a cDNA library. Theoligonucleotide sequences selected as probes should be of sufficientlength and sufficiently unambiguous that false positives are minimized.The oligonucleotide is preferably labeled such that it can be detectedupon hybridization to DNA in the library being screened. Methods oflabeling are well known in the art, and include the use of radiolabelslike ³²P-labeled ATP, biotinylation or enzyme labeling. Hybridizationconditions, including moderate stringency and high stringency, areprovided in Sambrook et al., supra.

Sequences identified in such library screening methods can be comparedand aligned to other known sequences deposited and available in publicdatabases such as GenBank or other private sequence databases. Sequenceidentity (at either the amino acid or nucleotide level) within definedregions of the molecule or across the full-length sequence can bedetermined using methods known in the art and as described herein.

Nucleic acid having protein coding sequence may be obtained by screeningselected cDNA or genomic libraries using the deduced amino acid sequencedisclosed herein for the first time, and, if necessary, usingconventional primer extension procedures as described in Sambrook etal., supra, to detect precursors and processing intermediates of mRNAthat may not have been reverse-transcribed into cDNA.

2. Selection and Transformation of Host Cells

Host cells are transfected or transformed with expression or cloningvectors described herein for PRO10282 production and cultured inconventional nutrient media modified as appropriate for inducingpromoters, selecting transformants, or amplifying the genes encoding thedesired sequences. The culture conditions, such as media, temperature,pH and the like, can be selected by the skilled artisan without undueexperimentation. In general, principles, protocols, and practicaltechniques for maximizing the productivity of cell cultures can be foundin Mammalian Cell Biotechnology: a Practical Approach, M. Butler, ed.(IRL Press, 1991) and Sambrook et al., supra.

Methods of eukaryotic cell transfection and prokaryotic celltransformation are known to the ordinarily skilled artisan, for example,CaCl₂, CaPO₄, liposome-mediated and electroporation. Depending on thehost cell used, transformation is performed using standard techniquesappropriate to such cells. The calcium treatment employing calciumchloride, as described in Sambrook et al., supra, or electroporation isgenerally used for prokaryotes. Infection with Agrobacterium tumefaciensis used for transformation of certain plant cells, as described by Shawet al., Gene, 23:315 (1983) and WO 89/05859 published 29 Jun. 1989. Formammalian cells without such cell walls, the calcium phosphateprecipitation method of Graham and van der Eb, Virology, 52:456-457(1978) can be employed. General aspects of mammalian cell host systemtransfections have been described in U.S. Pat. No. 4,399,216.Transformations into yeast are typically carried out according to themethod of Van Solingen et al., J. Bact., 130:946 (1977) and Hsiao etal., Proc. Natl. Acad. Sci. (USA), 76:3829 (1979). However, othermethods for introducing DNA into cells, such as by nuclearmicroinjection, electroporation, bacterial protoplast fusion with intactcells, or polycations, e.g., polybrene, polyornithine, may also be used.For various techniques for transforming mammalian cells, see Keown etal., Methods in Enzymology, 185:527-537 (1990) and Mansour et al.,Nature, 336:348-352 (1988).

Suitable host cells for cloning or expressing the DNA in the vectorsherein include prokaryote, yeast, or higher eukaryote cells. Suitableprokaryotes include but are not limited to eubacteria, such asGram-negative or Gram-positive organisms, for example,Enterobacteriaceae such as E. coli. Various E. coli strains are publiclyavailable, such as E. coli K12 strain MM294 (ATCC 31,446); E. coli X1776(ATCC 31,537); E. coli strain W3110 (ATCC 27,325) and K5 772 (ATCC53,635). Other suitable prokaryotic host cells includeEnterobacteriaceae such as Escherichia, e.g., E. coli, Enterobacter,Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium,Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacillisuch as B. subtilis and B. licheniformis (e.g., B. licheniformis 41Pdisclosed in DD 266,710 published 12 Apr. 1989), Pseudomonas such as P.aeruginosa, and Streptomyces. These examples are illustrative ratherthan limiting. Strain W3110 is one particularly preferred host or parenthost because it is a common host strain for recombinant DNA productfermentations. Preferably, the host cell secretes minimal amounts ofproteolytic enzymes. For example, strain W3110 may be modified to effecta genetic mutation in the genes encoding proteins endogenous to thehost, with examples of such hosts including E. coli W3110 strain 1A2,which has the complete genotype tonA; E. coli W3110 strain 9E4, whichhas the complete genotype tonA ptr3; E. coli W3110 strain 27C7 (ATCC55,244), which has the complete genotype tonA ptr3 phoA E15(argF-lac)169 degP ompT kan^(r) ; E. coli W3110 strain 37D6, which hasthe complete genotype tonA ptr3 phoA E15 (argF-lac)169 degP ompT rbs7ilvG kan^(r) ; E. coli W3110 strain 40B4, which is strain 37D6 with anon-kanamycin resistant degP deletion mutation; and an E. coli strainhaving mutant periplasmic protease disclosed in U.S. Pat. No. 4,946,783issued 7 Aug. 1990. Alternatively, in vitro methods of cloning, e.g.,PCR or other nucleic acid polymerase reactions, are suitable.

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable cloning or expression hosts forPRO10282-encoding vectors. Saccharomyces cerevisiae is a commonly usedlower eukaryotic host microorganism. Others include Schizosaccharomycespombe (Beach and Nurse, Nature, 290: 140 [1981]; EP 139,383 published 2May 1985); Kluyveromyces hosts (U.S. Pat. No. 4,943,529; Fleer et al.,Bio/Technology, 9:968-975 (1991)) such as, e.g., K. lactis (MW98-8C,CBS683, CBS4574; Louvencourt et al., J. Bacteriol., 154(2): 737-1742[1983]), K. fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K.vickeramii (ATCC 24,178), K. waltii (ATCC 56,500), K. drosophilarum(ATCC 36,906; Van den Berg et al., Bio/Technology, 8:135 (1990)), K.thermotolerans, and K. marxianus; yarrowia (EP 402,226); Pichia pastoris(EP 183,070; Sreekrishna et al., J. Basic Microbiol., 28:265-278[1988]); Candida; Tricioderma reesia (EP 244,234); Neurospora crassa(Case et al., Proc. Natl. Acad. Sci. USA, 76:5259-5263 [1979]);Schwanniomyces such as Schwanniomyces occidentalis (EP 394,538 published31 Oct. 1990); and filamentous fungi such as, e.g., Neurospora,Penicillium, Tolypocladium (WO 91/00357 published 10 Jan. 1991), andAspergillus hosts such as A. nidulans (Ballance et al., Biochem.Biophys. Res. Commun., 112:284-289 [1983]; Tilburn et al., Gene,26:205-221 [1983]; Yelton et al., Proc. Natl. Acad. Sci. USA, 81:1470-1474 [1984]) and A. niger (Kelly and Hynes, EMBO J., 4:475-479[1985]). Methylotropic yeasts are suitable herein and include, but arenot limited to, yeast capable of growth on methanol selected from thegenera consisting of Hansenula, Candida, Kloeckera, Pichia,Saccharomyces, Torulopsis, and Rhodotorula. A list of specific speciesthat are exemplary of this class of yeasts may be found in C. Anthony,The Biochemistry of Methylotrophs, 269 (1982).

Suitable host cells for the expression of glycosylated PRO10282 arederived from multicellular organisms. Examples of invertebrate cellsinclude insect cells such as Drosophila S2 and Spodoptera Sf9, as wellas plant cells. Examples of useful mammalian host cell lines includeChinese hamster ovary (CHO) and COS cells. More specific examplesinclude monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL1651); human embryonic kidney line (293 or 293 cells subcloned forgrowth in suspension culture, Graham et al., J. Gen Virol., 36:59(1977)); Chinese hamster ovary cells/-DHFR (CHO, Urlaub and Chasin,Proc. Natl. Acad. Sci. USA, 77:4216 (1980)); mouse sertoli cells (TM4,Mather, Biol. Reprod., 23:243-251 (1980)); human lung cells (W138, ATCCCCL 75); human liver cells (Hep G2, HB 8065); and mouse mammary tumor(MMT 060562, ATCC CCL51). The selection of the appropriate host cell isdeemed to be within the skill in the art.

3. Selection and Use of a Replicable Vector

The nucleic acid (e.g., cDNA or genomic DNA) encoding a PRO10282 may beinserted into a replicable vector for cloning (amplification of the DNA)or for expression. Various vectors are publicly available. The vectormay, for example, be in the form of a plasmid, cosmid, viral particle,or phage. The appropriate nucleic acid sequence may be inserted into thevector by a variety of procedures. In general, DNA is inserted into anappropriate restriction endonuclease site(s) using techniques known inthe art. Vector components generally include, but are not limited to,one or more of a signal sequence, an origin of replication, one or moremarker genes, an enhancer element, a promoter, and a transcriptiontermination sequence. Construction of suitable vectors containing one ormore of these components employs standard ligation techniques which areknown to the skilled artisan.

The PRO10282 may be produced recombinantly not only directly, but alsoas a fusion polypeptide with a heterologous polypeptide, which may be asignal sequence or other polypeptide having a specific cleavage site atthe N-terminus of the mature protein or polypeptide. In general, thesignal sequence may be a component of the vector, or it may be a part ofthe PRO10282-encoding DNA that is inserted into the vector. The signalsequence may be a prokaryotic signal sequence selected, for example,from the group of the alkaline phosphatase, penicillinase, lpp, orheat-stable enterotoxin H leaders. For yeast secretion the signalsequence may be, e.g., the yeast invertase leader, alpha factor leader(including Saccharomyces and Kluyveromyces—factor leaders, the latterdescribed in U.S. Pat. No. 5,010,182), or acid phosphatase leader, theC. albicans glucoamylase leader (EP 362,179 published 4 Apr. 1990), orthe signal described in WO 90/13646 published 15 Nov. 1990. In mammaliancell expression, mammalian signal sequences may be used to directsecretion of the protein, such as signal sequences from secretedpolypeptides of the same or related species, as well as viral secretoryleaders.

Both expression and cloning vectors contain a nucleic acid sequence thatenables the vector to replicate in one or more selected host cells. Suchsequences are well known for a variety of bacteria, yeast, and viruses.The origin of replication from the plasmid pBR322 is suitable for mostGram-negative bacteria, the 2 plasmid origin is suitable for yeast, andvarious viral origins (SV40, polyoma, adenovirus, VSV or BPV) are usefulfor cloning vectors in mammalian cells.

Expression and cloning vectors will typically contain a selection gene,also termed a selectable marker. Typical selection genes encode proteinsthat (a) confer resistance to antibiotics or other toxins, e.g.,ampicillin, neomycin, methotrexate, or tetracycline, (b) complementauxotrophic deficiencies, or (c) supply critical nutrients not availablefrom complex media, e.g., the gene encoding D-alanine racemase forBacilli.

An example of suitable selectable markers for mammalian cells are thosethat enable the identification of cells competent to take up thePRO10282-encoding nucleic acid, such as DHFR or thymidine kinase. Anappropriate host cell when wild-type DHFR is employed is the CHO cellline deficient in DHFR activity, prepared and propagated as described byUrlaub et al., Proc. Natl. Acad. Sci. USA, 77:4216 (1980). A suitableselection gene for use in yeast is the trp1 gene present in the yeastplasmid YRp7 [Stinchcomb et al., Nature, 282:39 (1979); Kingsman et al.,Gene, 7:141 (1979); Tschemper et al., Gene, 10:157 (1980)]. The trp1gene provides a selection marker for a mutant strain of yeast lackingthe ability to grow in tryptophan, for example, ATCC No. 44076 or PEP4-1[Jones, Genetics, 85:12 (1977)].

Expression and cloning vectors usually contain a promoter operablylinked to the PRO10282-encoding nucleic acid sequence to direct mRNAsynthesis. Promoters recognized by a variety of potential host cells arewell known. Promoters suitable for use with prokaryotic hosts includethe -lactamase and lactose promoter systems [Chang et al., Nature,275:615 (1978); Goeddel et al., Nature, 281:544 (1979)], alkalinephosphatase, a tryptophan (trp) promoter system [Goeddel, Nucleic AcidsRes., 8:4057 (1980); EP 36,776], and hybrid promoters such as the tacpromoter [deBoer et al., Proc. Natl. Acad. Sci. USA, 80:21-25 (1983)].Promoters for use in bacterial systems also will contain aShine-Dalgarno (S.D.) sequence operably linked to the DNA encodingPRO10282.

Examples of suitable promoting sequences for use with yeast hostsinclude the promoters for 3-phosphoglycerate kinase [Hitzeman et al., J.Biol. Chem., 255:2073 (1980)] or other glycolytic enzymes [Hess et al.,J. Adv. Enzyme Reg., 7:149 (1968); Holland, Biochemistry, 17:4900(1978)], such as enolase, glyceraldehyde-3-phosphate dehydrogenase,hexokinase, pyruvate decarboxylase, phosphofructokinase,glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvatekinase, triosephosphate isomerase, phosphoglucose isomerase, andglucokinase.

Other yeast promoters, which are inducible promoters having theadditional advantage of transcription controlled by growth conditions,are the promoter regions for alcohol dehydrogenase 2, isocytochrome C,acid phosphatase, degradative enzymes associated with nitrogenmetabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase,and enzymes responsible for maltose and galactose utilization. Suitablevectors and promoters for use in yeast expression are further describedin EP 73,657.

PRO10282 transcription from vectors in mammalian host cells iscontrolled, for example, by promoters obtained from the genomes ofviruses such as polyoma virus, fowlpox virus (UK 2,211,504 published 5Jul. 1989), adenovirus (such as Adenovirus 2), bovine papilloma virus,avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-B virusand Simian Virus 40 (SV40), from heterologous mammalian promoters, e.g.,the actin promoter or an immunoglobulin promoter, and from heat-shockpromoters, provided such promoters are compatible with the host cellsystems.

Transcription of a DNA encoding the PRO10282 by higher eukaryotes may beincreased by inserting an enhancer sequence into the vector. Enhancersare cis-acting elements of DNA, usually about from 10 to 300 bp, thatact on a promoter to increase its transcription. Many enhancer sequencesare now known from mammalian genes (globin, elastase, albumin,-fetoprotein, and insulin). Typically, however, one will use an enhancerfrom a eukaryotic cell virus. Examples include the SV40 enhancer on thelate side of the replication origin (bp 100-270), the cytomegalovirusearly promoter enhancer, the polyoma enhancer on the late side of thereplication origin, and adenovirus enhancers. The enhancer may bespliced into the vector at a position 5′ or 3′ to the PRO10282 codingsequence, but is preferably located at a site 5′ from the promoter.

Expression vectors used in eukaryotic host cells (yeast, fungi, insect,plant, animal, human, or nucleated cells from other multicellularorganisms) will also contain sequences necessary for the termination oftranscription and for stabilizing the mRNA. Such sequences are commonlyavailable from the 5′ and, occasionally 3′, untranslated regions ofeukaryotic or viral DNAs or cDNAs. These regions contain nucleotidesegments transcribed as polyadenylated fragments in the untranslatedportion of the mRNA encoding PRO10282.

Still other methods, vectors, and host cells suitable for adaptation tothe synthesis of PRO10282 in recombinant vertebrate cell culture aredescribed in Gething et al., Nature, 293:620-625 (1981); Mantei et al.,Nature, 281:40-46 (1979); EP 117,060; and EP 117,058.

4. Detecting Gene Amplification/Expression

Gene amplification and/or expression may be measured in a sampledirectly, for example, by conventional Southern blotting, Northernblotting to quantitate the transcription of mRNA [Thomas, Proc. Natl.Acad. Sci. USA, 77:5201-5205 (1980)], dot blotting (DNA analysis), or insitu hybridization, using an appropriately labeled probe, based on thesequences provided herein. Alternatively, antibodies may be employedthat can recognize specific duplexes, including DNA duplexes, RNAduplexes, and DNA-RNA hybrid duplexes or DNA-protein duplexes. Theantibodies in turn may be labeled and the assay may be carried out wherethe duplex is bound to a surface, so that upon the formation of duplexon the surface, the presence of antibody bound to the duplex can bedetected.

Gene expression, alternatively, may be measured by immunologicalmethods, such as immunohistochemical staining of cells or tissuesections and assay of cell culture or body fluids, to quantitatedirectly the expression of gene product. Antibodies useful forimmunohistochemical staining and/or assay of sample fluids may be eithermonoclonal or polyclonal, and may be prepared in any mammal.Conveniently, the antibodies may be prepared against a native sequencePRO10282 polypeptide or against a synthetic peptide based on the DNAsequences provided herein or against exogenous sequence fused toPRO10282 DNA and encoding a specific antibody epitope.

5. Purification of Polypeptide

Forms of PRO10282 may be recovered from culture medium or from host celllysates. If membrane-bound, it can be released from the membrane using asuitable detergent solution (e.g. Triton-X 100) or by enzymaticcleavage. Cells employed in expression of PRO10282 can be disrupted byvarious physical or chemical means, such as freeze-thaw cycling,sonication, mechanical disruption, or cell lysing agents.

It may be desired to purify PRO10282 from recombinant cell proteins orpolypeptides. The following procedures are exemplary of suitablepurification procedures: by fractionation on an ion-exchange column;ethanol precipitation; reverse phase HPLC; chromatography on silica oron a cation-exchange resin such as DEAE; chromatofocusing; SDS-PAGE;ammonium sulfate precipitation; gel filtration using, for example,Sephadex G-75; protein A Sepharose columns to remove contaminants suchas IgG; and metal chelating columns to bind epitope-tagged forms of thePRO10282. Various methods of protein purification may be employed andsuch methods are known in the art and described for example inDeutscher, Methods in Enzymology, 182 (1990); Scopes, ProteinPurification: Principles and Practice, Springer-Verlag, New York (1982).The purification step(s) selected will depend, for example, on thenature of the production process used and the particular PRO10282produced.

E. Amplification of Genes Encoding the PRO10282 (Stra6) Polypeptides inTumor Tissues and Cell Lines

In one aspect, the present invention is based on the identification andcharacterization of genes that are amplified in certain cancer cells.

The genome of prokaryotic and eukaryotic organisms is subjected to twoseemingly conflicting requirements. One is the preservation andpropagation of DNA as the genetic information in its original form, toguarantee stable inheritance through multiple generations. On the otherhand, cells or organisms must be able to adapt to lasting environmentalchanges. The adaptive mechanisms can include qualitative or quantitativemodifications of the genetic material. Qualitative modifications includeDNA mutations, in which coding sequences are altered resulting in astructurally and/or functionally different protein. Gene amplificationis a quantitative modification, whereby the actual number of completecoding sequence, i.e., a gene, increases, leading to an increased numberof available templates for transcription, an increased number oftranslatable transcripts, and, ultimately, to an increased abundance ofthe protein encoded by the amplified gene.

The phenomenon of gene amplification and its underlying mechanisms havebeen investigated in vitro in several prokaryotic and eukaryotic culturesystems. The best-characterized example of gene amplification involvesthe culture of eukaryotic cells in medium containing variableconcentrations of the cytotoxic drug methotrexate (MTX). MTX is a folicacid analogue and interferes with DNA synthesis by blocking the enzymedihydrofolate reductase (DHFR). During the initial exposure to lowconcentrations of MTX most cells (>99.9%) will die. A small number ofcells survive, and are capable of growing in increasing concentrationsof MTX by producing large amounts of DHFR-RNA and protein. The basis ofthis overproduction is the amplification of the single DHFR gene. Theadditional copies of the gene are found as extrachromosomal copies inthe form of small, supernumerary chromosomes (double minutes) or asintegrated chromosomal copies.

Gene amplification is most commonly encountered in the development ofresistance to cytotoxic drugs (antibiotics for bacteria andchemotherapeutic agents for eukaryotic cells) and neoplastictransformation. Transformation of a eukaryotic cell as a spontaneousevent or due to a viral or chemical/environmental insult is typicallyassociated with changes in the genetic material of that cell. One of themost common genetic changes—observed in human malignancies are mutationsof the p53 protein. p53 controls the transition of cells from thestationary (G1) to the replicative (S) phase and prevents thistransition in the presence of DNA damage. In other words, one of themain consequences of disabling p53 mutations is the accumulation andpropagation of DNA damage, i.e., genetic changes. Common types ofgenetic changes in neoplastic cells are, in addition to point mutations,amplifications and gross, structural alterations, such astranslocations.

The amplification of DNA sequences may indicate a specific functionalrequirement as illustrated in the DHFR experimental system. Therefore,the amplification of certain oncogenes in malignancies points toward acausative role of these genes in the process of malignant transformationand maintenance of the transformed phenotype. This hypothesis has gainedsupport in recent studies. For example, the bcl-2 protein was found tobe amplified in certain types of non-Hodgkin's lymphoma. This proteininhibits apoptosis and leads to the progressive accumulation ofneoplastic cells. Members of the gene family of growth factor receptorshave been found to be amplified in various types of cancers suggestingthat overexpression of these receptors may make neoplastic cells lesssusceptible to limiting amounts of available growth factor. Examplesinclude the amplification of the androgen receptor in recurrent prostatecancer during androgen deprivation therapy and the amplification of thegrowth factor receptor homologue ERB2 in breast cancer. Lastly, genesinvolved in intracellular signaling and control of cell cycleprogression can undergo amplification during malignant transformation.This is illustrated by the amplification of the bcl-1 and ras genes invarious epithelial and lymphoid neoplasms.

These earlier studies illustrate the feasibility of identifyingamplified DNA sequences in neoplasms, because this approach can identifygenes important for malignant transformation. The case of ERB2 alsodemonstrates the feasibility from a therapeutic standpoint, sincetransforming proteins may represent novel and specific targets for tumortherapy.

Several different techniques can be used to demonstrate amplifiedgenomic sequences. Classical cytogenetic analysis of chromosome spreadsprepared from cancer cells is adequate to identify gross structuralalterations, such as translocations, deletions and inversions. Amplifiedgenomic regions can only be visualized, if they involve large regionswith high copy numbers or are present as extrachromosomal material.While cytogenetics was the first technique to demonstrate the consistentassociation of specific chromosomal changes with particular neoplasms,it is inadequate for the identification and isolation of manageable DNAsequences. The more recently developed technique of comparative genomichybridization (CGH) has illustrated the widespread phenomenon of genomicamplification in neoplasms. Tumor and normal DNA are hybridizedsimultaneously onto metaphases of normal cells and the entire genome canbe screened by image analysis for DNA sequences that are present in thetumor at an increased frequency. (WO 93/18,186; Gray et al., RadiationRes., 137:275-289 [1994]). As a screening method, this type of analysishas revealed a large number of recurring amplicons (a stretch ofamplified DNA) in a variety of human neoplasms. Although CGH is moresensitive than classical cytogenetic analysis in identifying amplifiedstretches of DNA, it does not allow a rapid identification and isolationof coding sequences within the amplicon by standard molecular genetictechniques.

The most sensitive methods to detect gene amplification are polymerasechain reaction (PCR)-based assays. These assays utilize very smallamount of tumor DNA as starting material, are exquisitely sensitive,provide DNA that is amenable to further analysis, such as sequencing andare suitable for high-volume throughput analysis.

The above-mentioned assays are not mutually exclusive, but arefrequently used in combination to identify amplifications in neoplasms.While cytogenetic analysis and CGH represent screening methods to surveythe entire genome for amplified regions, PCR-based assays are mostsuitable for the final identification of coding sequences, i.e., genesin amplified regions.

According to the present invention, such genes have been identified byquantitative PCR (S. Gelmini et al., Clin. Chem. 43:752 [1997]), bycomparing RNA from a variety of primary tumors, including breast lung,colon, prostate, brain, liver, kidney, pancrease, spleen, thymus,testis, ovary, uterus, etc., tumor, or tumor cell lines, with pooled DNAfrom healthy donors. Quantitative PCR was performed using a TaqManinstrument (ABI). Gene-specific primers and fluorogenic probes weredesigned based upon the coding sequences of the DNAs.

Human lung carcinoma cell lines include A549 (SRCC768), Calu-1(SRCC769), Calu-6 (SRCC770), H157 (SRCC771), H441 (SRCC772), H460(SRCC773), SKMES-1 (SRCC774), SW900 (SRCC775), H522 (SRCC832), and H810(SRCC833), all available from ATCC. Primary human lung tumor cellsusually derive from adenocarcinomas, squamous cell carcinomas, largecell carcinomas, non-small cell carcinomas, small cell carcinomas, andbroncho alveolar carcinomas, and include, for example, SRCC724(adenocarcinoma, abbreviated as “AdenoCa”)(LT1), SRCC725 (squamous cellcarcinoma, abbreviated as “SqCCa)(LT1a), SRCC726 (adenocarcinoma)(LT2),SRCC727 (adenocarcinoma)(LT3), SRCC728 (adenocarcinoma)(LT4), SRCC729(squamous cell carcinoma)(LT6), SRCC730 (adeno/squamous cellcarcinoma)(LT7), SRCC731 (adenocarcinoma)(LT9), SRCC732 (squamous cellcarcinoma)(LT10), SRCC733 (squamous cell carcinoma)(LT11), SRCC734(adenocarcinoma)(LT12), SRCC735 (adeno/squamous cell carcinoma)(LT13),SRCC736 (squamous cell carcinoma)(LT15), SRCC737 (squamous cellcarcinoma)(LT16), SRCC738 (squamous cell carcinoma)(LT17), SRCC739(squamous cell carcinoma)(LT18), SRCC740 (squamous cellcarcinoma)(LT19), SRCC741 (lung cell carcinoma, abbreviated as“LCCa”)(LT21), SRCC811 (adenocarcinoma)(LT22), SRCC825(adenocarcinoma)(LT8), SRCC886 (adenocarcinoma)(LT25), SRCC887 (squamouscell carcinoma)(LT26), SRCC888 (adeno-BAC carcinoma)(LT27), SRCC889(squamous cell carcinoma)(LT28), SRCC890 (squamous cellcarcinoma)(LT29), SRCC891 (adenocarcinoma)(LT30), SRCC892 (squamous cellcarcinoma)(LT31), SRCC894 (adenocarcinoma)(LT33). Also included arehuman lung tumors designated SRCC1125 [HF-000631], SRCC1127 [HF-000641],SRCC1129 [HF-000643], SRCC1133 [HF-000840], SRCC1135 [HF-000842],SRCC1227 [HF-001291], SRCC1229 [HF-001293], SRCC1230 [HF-001294],SRCC1231 [HF-001295], SRCC1232 [HF-001296], SRCC1233 [HF-001297],SRCC1235 [HF-001299], and SRCC1236 [HF-001300].

Colon cancer cell lines include, for example, ATCC cell lines SW480(adenocarcinoma, SRCC776), SW620 (lymph node metastasis of colonadenocarcinoma, SRCC777), Colo320 (carcinoma, SRCC778), HT29(adenocarcinoma, SRCC779), HM7 (a high mucin producing variant of ATCCcolon adenocarcinoma cell line. SRCC780, obtained from Dr. RobertWarren, UCSF), CaWiDr (adenocarcinoma, SRCC781), HCT116 (carcinoma,SRCC782), SKCO1 (adenocarcinoma, SRCC783), SW403 (adenocarcinoma,SRCC784), LS174T (carcinoma, SRCC785), Colo205 (carcinoma, SRCC828),HCT15 (carcinoma, SRCC829), HCC2998 (carcinoma, SRCC830), and KM12(carcinoma, SRCC831). Primary colon tumors include colon adenocarcinomasdesignated CT2 (SRCC742), CT3 (SRCC743), CT8 (SRCC744), CT10(SRCC745),CT12 (SRCC746), CT14 (SRCC747), CT15 (SRCC748), CT16 (SRCC749), CT17(SRCC750), CT1 (SRCC751), CT4 (SRCC752), CT5 (SRCC753), CT6 (SRCC754),CT7 (SRCC755), CT9 (SRCC756), CT11 (SRCC757), CT18 (SRCC758), CT19(adenocarcinoma, SRCC906), CT20 (adenocarcinoma, SRCC907), CT21(adenocarcinoma, SRCC908), CT22 (adenocarcinoma, SRCC909), CT23(adenocarcinoma, SRCC910), CT24 (adenocarcinoma, SRCC911), CT25(adenocarcinoma, SRCC912), CT26 (adenocarcinoma, SRCC913), CT27(adenocarcinoma, SRCC914), CT28 (adenocarcinoma, SRCC915), CT29(adenocarcinoma, SRCC916), CT30 (adenocarcinoma, SRCC917), CT31(adenocarcinoma, SRCC918), CT32 (adenocarcinoma, SRCC919), CT33(adenocarcinoma, SRCC920), CT35 (adenocarcinoma, SRCC921), and CT36(adenocarcinoma, SRCC922). Also included are human colon tumorsdesignated SRCC1051 [HF-000499], SRCC1052 [HF-000539], SRCC1053[HF-000575], SRCC1054 [HF-000698], SRCC1142 [HF-000762], SRCC1144[HF-000789], SRCC1146 [HF-000795] and SRCC1148-[HF-000811].

Human breast carcinoma cell lines include, for example, HBL100(SRCC759),MB435s (SRCC760), T47D (SRCC761), MB468 (SRCC762), MB175 (SRCC763),MB361 (SRCC764), BT20 (SRCC765), MCF7 (SRCC766), and SKBR3 (SRCC767),and human breast tumor center designated SRCC1057 [HF-000545]. Alsoincluded are human breast tumors designated SRCC1094, SRCC1095,SRCC1096, SRCC1097, SRCC1098, SRCC1099, SRCC1100, SRCC1101, and humanbreast-met-lung-NS tumor designated SRCC893 [LT 32].

Human kidney tumor centers include SRCC989 [HF-000611] and SRCC1014[HF-000613].

Human testis tumor center includes SRCC1001 [HF-000733] and testis tumormargin SRCC999 [HF-000716].

Human parathyroid tumor includes SRCC1002 [HF-000831] and SRCC1003[HF-000832].

F. Tissue Distribution

The results of the gene amplification assays herein can be verified byfurther studies, such as, by determining mRNA expression in varioushuman tissues.

As noted before, gene amplification and/or gene expression in varioustissues may be measured by conventional Southern blotting, Northernblotting to quantitate the transcription of mRNA (Thomas, Proc. Natl.Acad. Sci. USA, 77:5201-5205 [1980]), dot blotting (DNA analysis), or insitu hybridization, using an appropriately labeled probe, based on thesequences provided herein. Alternatively, antibodies may be employedthat can recognize specific duplexes, including DNA duplexes, RNAduplexes, and DNA-RNA hybrid duplexes or DNA-protein duplexes.

Gene expression in various tissues, alternatively, may be measured byimmunological methods, such as immunohistochemical staining of tissuesections and assay of cell culture or body fluids, to quantitatedirectly the expression of gene product. Antibodies useful forimmunohistochemical staining and/or assay of sample fluids may be eithermonoclonal or polyclonal, and may be prepared in any mammal.Conveniently, the antibodies may be prepared against a native sequencePRO10282 (Stra6) polypeptide or against a synthetic peptide based on theDNA and encoding a specific antibody epitope. General techniques forgenerating antibodies, and special protocols for Northern blotting andin situ hybridization are provided hereinbelow.

G. Chromosome Mapping

If the amplification of a given gene is functionally relevant, then thatgene should be amplified more than neighboring genomic regions which arenot important for tumor survival. To test this, the gene can be mappedto a particular chromosome, e.g., by radiation-hybrid analysis. Theamplification level is then determined at the location identified, andat the neighboring genomic region. Selective or preferentialamplification at the genomic region to which the gene has been mapped isconsistent with the possibility that the gene amplification observedpromotes tumor growth or survival. Chromosome mapping includes bothframework and epicenter mapping. For further details see, e.g., Stewartet al., Genome Research, 7:422-433 (1997).

H. Antibody Binding Studies

The results of the gene amplification study can be further verified byantibody binding studies, in which the ability of anti-PRO10282(anti-Stra6) antibodies to inhibit the expression of Stra6 polypeptideson tumor (cancer) cells is tested. Exemplary antibodies includepolyclonal, monoclonal, humanized, bispecific, and heteroconjugateantibodies, the preparation of which will be described hereinbelow.

Antibody binding studies may be carried out in any known assay method,such as competitive binding assays, direct and indirect sandwich assays,and immunoprecipitation assays. Zola, Monoclonal Antibodies: A Manual ofTechniques, pp. 147-158 (CRC Press, Inc., 1987).

Competitive binding assays rely on the ability of a labeled standard tocompete with the test sample analyte for binding with a limited amountof antibody. The amount of target protein (encoded by a gene amplifiedin a tumor cell) in the test sample is inversely proportional to theamount of standard that becomes bound to the antibodies. To facilitatedetermining the amount of standard that becomes bound, the antibodiespreferably are insolubilized before or after the competition, so thatthe standard and analyte that are bound to the antibodies mayconveniently be separated from the standard and analyte which remainunbound.

Sandwich assays involve the use of two antibodies, each capable ofbinding to a different immunogenic portion, or epitope, of the proteinto be detected. In a sandwich assay, the test sample analyte is bound bya first antibody which is immobilized on a solid support, and thereaftera second antibody binds to the analyte, thus forming an insolublethree-part complex. See, e.g., U.S. Pat. No. 4,376,110. The secondantibody may itself be labeled with a detectable moiety (direct sandwichassays) or may be measured using an anti-immunoglobulin antibody that islabeled with a detectable moiety (indirect sandwich assay). For example,one type of sandwich assay is an ELISA assay, in which case thedetectable moiety is an enzyme.

For immunohistochemistry, the tumor sample may be fresh or frozen or maybe embedded in paraffin and fixed with a preservative such as formalin,for example.

I. Cell-Based Tumor Assays

Cell-based assays and animal models for tumors (e.g., cancers) can beused to verify the findings of the gene amplification assay, and furtherunderstand the relationship between the genes identified herein and thedevelopment and pathogenesis of neoplastic cell growth. The role of geneproducts identified herein in the development and pathology of tumor orcancer can be tested by using primary tumor cells or cells lines thathave been identified to amplify the genes herein. Such cells include,for example, the breast, colon and lung cancer cells and cell lineslisted above.

In a different approach, cells of a cell type known to be involved in aparticular tumor are transfected with the cDNAs herein, and the abilityof these cDNAs to induce excessive growth is analyzed. Suitable cellsinclude, for example, stable tumor cells lines such as, the B104-1-1cell line (stable NIH-3T3 cell line transfected with the neuprotooncogene) and ras-transfected NIH-3T3 cells, which can betransfected with the desired gene, and monitored for tumorogenic growth.Such transfected cell lines can then be used to test the ability ofpoly- or monoclonal antibodies or antibody compositions to inhibittumorogenic cell growth by exerting cytostatic or cytotoxic activity onthe growth of the transformed cells, or by mediating antibody-dependentcellular cytotoxicity (ADCC). Cells transfected with the codingsequences of the genes identified herein can further be used to identifydrug candidates for the treatment of cancer.

In addition, primary cultures derived from tumors in transgenic animals(as described below) can be used in the cell-based assays herein,although stable cell lines are preferred. Techniques to derivecontinuous cell lines from transgenic animals are well known in the art(see, e.g., Small et al., Mol. Cell. Biol., 5:642-648 [1985]).

J. Animal Models

A variety of well known animal models can be used to further understandthe role of the genes identified herein in the development andpathogenesis of tumors, and to test the efficacy of candidatetherapeutic agents, including antibodies, and other antagonists of thenative polypeptides, including small molecule antagonists. The in vivonature of such models makes them particularly predictive of responses inhuman patients. Animal models of tumors and cancers (e.g., breastcancer, colon cancer, prostate cancer, lung cancer, etc.) include bothnon-recombinant and recombinant (transgenic) animals. Non-recombinantanimal models include, for example, rodent, e.g., murine models. Suchmodels can be generated by introducing tumor cells into syngeneic miceusing standard techniques, e.g., subcutaneous injection, tail veininjection, spleen implantation, intraperitoneal implantation,implantation under the renal capsule, or orthopin implantation, e.g.,colon cancer cells implanted in colonic tissue. (See, e.g., PCTpublication No. WO 97/33551, published Sep. 18, 1997).

Probably the most often used animal species in oncological studies areimmunodeficient mice and, in particular, nude mice. The observation thatthe nude mouse with hypo/aplasia could successfully act as a host forhuman tumor xenografts has lead to its widespread use for this purpose.The autosomal recessive nu gene has been introduced into a very largenumber of distinct congenic strains of nude mouse, including, forexample, ASW, A/He, AKR, BALB/c, B10.LP, C17, C3H, C57BL, C57, CBA, DBA,DDD, I/st, NC, NFR, NFS, NFS/N, NZB, NZC, NZW, P, RIII and SJL. Inaddition, a wide variety of other animals with inherited immunologicaldefects other than the nude mouse have been bred and used as recipientsof tumor xenografts. For further details see, e.g., The Nude Mouse inOncology Research, E. Boven and B. Winograd, eds., CRC Press, Inc.,1991.

The cells introduced into such animals can be derived from knowntumor/cancer cell lines, such as, any of the above-listed tumor celllines, and, for example, the B104-1-1 cell line (stable NIH-3T3 cellline transfected with the neu protooncogene); ras-transfected NIH-3T3cells; Caco-2 (ATCC HTB-37); a moderately well-differentiated grade IIhuman colon adenocarcinoma cell line, HT-29 (ATCC HTB-38), or fromtumors and cancers. Samples of tumor or cancer cells can be obtainedfrom patients undergoing surgery, using standard conditions, involvingfreezing and storing in liquid nitrogen (Karmali et al., Br. J. Cancer,48:689-696 [1983]).

Tumor cells can be introduced into animals, such as nude mice, by avariety of procedures. The subcutaneous (s.c.) space in mice is verysuitable for tumor implantation. Tumors can be transplanted s.c. assolid blocks, as needle biopsies by use of a trochar, or as cellsuspensions. For solid block or trochar implantation, tumor tissuefragments of suitable size are introduced into the s.c. space. Cellsuspensions are freshly prepared from primary tumors or stable tumorcell lines, and injected subcutaneously. Tumor cells can also beinjected as subdermal implants. In this location, the inoculum isdeposited between the lower part of the dermal connective tissue and thes.c. tissue. Boven and Winograd (1991), supra.

Animal models of breast cancer can be generated, for example, byimplanting rat neuroblastoma cells (from which the neu oncogen wasinitially isolated), or neu-transformed NIH-3T3 cells into nude mice,essentially as described by Drebin et al., PNAS USA, 83:9129-9133(1986).

Similarly, animal models of colon cancer can be generated by passagingcolon cancer cells in animals, e.g., nude mice, leading to theappearance of tumors in these animals. An orthotopic transplant model ofhuman colon cancer in nude mice has been described, for example, by Wanget al., Cancer Research, 54:4726-4728 (1994) and Too et al., CancerResearch, 55:681-684 (1995). This model is based on the so-called“METAMOUSE” sold by AntiCancer, Inc., (San Diego, Calif.).

Tumors that arise in animals can be removed and cultured in vitro. Cellsfrom the in vitro cultures can then be passaged to animals. Such tumorscan serve as targets for further testing or drug screening.Alternatively, the tumors resulting from the passage can be isolated andRNA from pre-passage cells and cells isolated after one or more roundsof passage analyzed for differential expression of genes of interest.Such passaging techniques can be performed with any known tumor orcancer cell lines.

For example, Meth A, CMS4, CMS5, CMS21, and WEHI-164 are chemicallyinduced fibrosarcomas of BALB/c female mice (DeLeo et al., J. Exp. Med.,146:720 1977]), which provide a highly controllable model system forstudying the anti-tumor activities of various agents (Palladino et al.,J. Immunol., 138:4023-4032 [1987]). Briefly, tumor cells are propagatedin vitro in cell culture. Prior to injection into the animals, the celllines are washed and suspended in buffer, at a cell density of about10×10⁶ to 10×10⁷ cells/ml. The animals are then infected subcutaneouslywith 10 to 100 μl of the cell suspension, allowing one to three weeksfor a tumor to appear.

In addition, the Lewis lung (3LL) carcinoma of mice, which is one of themost thoroughly studied experimental tumors, can be used as aninvestigational tumor model. Efficacy in this tumor model has beencorrelated with beneficial effects in the treatment of human patientsdiagnosed with small cell carcinoma of the lung (SCCL). This tumor canbe introduced in normal mice upon injection of tumor fragments from anaffected mouse or of cells maintained in culture (Zupi et al., Br. J.Cancer, 41: suppl. 4:309 [1980]), and evidence indicates that tumors canbe started from injection of even a single cell and that a very highproportion of infected tumor cells survive. For further informationabout this tumor model see, Zacharski, Haemostasis, 16:300-320 [1986]).

One way of evaluating the efficacy of a test compound in an animal modelon an implanted tumor is to measure the size of the tumor before andafter treatment. Traditionally, the size of implanted tumors has beenmeasured with a slide caliper in two or three dimensions. The measurelimited to two dimensions does not accurately reflect the size of thetumor, therefore, it is usually converted into the corresponding volumeby using a mathematical formula. However, the measurement of tumor sizeis very inaccurate. The therapeutic effects of a drug candidate can bebetter described as treatment-induced growth delay and specific growthdelay. Another important variable in the description of tumor growth isthe tumor volume doubling time. Computer programs for the calculationand description of tumor growth are also available, such as the programreported by Rygaard and Spang-Thomsen, Proc. 6th Int. Workshop onImmune-Deficient Animals, Wu and Sheng eds., Basel, 1989, 301. It isnoted, however, that necrosis and inflammatory responses followingtreatment may actually result in an increase in tumor size, at leastinitially. Therefore, these changes need to be carefully monitored, by acombination of a morphometric method and flow cytometric analysis.

Recombinant (transgenic) animal models can be engineered by introducingthe coding portion of the genes identified herein into the genome ofanimals of interest, using standard techniques for producing transgenicanimals. Animals that can serve as a target for transgenic manipulationinclude, without limitation, mice, rats, rabbits, guinea pigs, sheep,goats, pigs, and non-human primates, e.g., baboons, chimpanzees andmonkeys. Techniques known in the art to introduce a transgene into suchanimals include pronucleic microinjection (Hoppe and Wanger, U.S. Pat.No. 4,873,191); retrovirus-mediated gene transfer into germ lines (e.g.,Van der Putten et al., Proc. Natl. Acad. Sci. USA, 82:6148-615 [1985]);gene targeting in embryonic stem cells (Thompson et al., Cell,56:313-321 [1989]); electroporation of embryos (Lo, Mol. Cell. Biol.,3:1803-1814 [1983]); sperm-mediated gene transfer (Lavitrano et al.,Cell, 57:717-73 [1989]). For review, see, for example, U.S. Pat. No.4,736,866.

For the purpose of the present invention, transgenic animals includethose that carry the transgene only in part of their cells (“mosaicanimals”). The transgene can be integrated either as a single transgene,or in concatamers, e.g., head-to-head or head-to-tail tandems. Selectiveintroduction of a transgene into a particular cell type is also possibleby following, for example, the technique of Lasko et al., Proc. Natl.Acad. Sci. USA, 89:6232-636 (1992).

The expression of the transgene in transgenic animals can be monitoredby standard techniques. For example, Southern blot analysis or PCRamplification can be used to verify the integration of the transgene.The level of mRNA expression can then be analyzed using techniques suchas in situ hybridization, Northern blot analysis, PCR, orimmunocytochemistry. The animals are further examined for signs of tumoror cancer development.

Alternatively, “knock out” animals can be constructed which have adefective or altered gene encoding a PRO10282 (Stra6) polypeptideidentified herein, as a result of homologous recombination between theendogenous gene encoding the polypeptide and altered genomic DNAencoding the same polypeptide introduced into an embryonic cell of theanimal. For example, cDNA encoding a PRO10282 polypeptide can be used toclone genomic DNA encoding that polypeptide in accordance withestablished techniques. A portion of the genomic DNA encoding aparticular PRO10282 polypeptide can be deleted or replaced with anothergene, such as a gene encoding a selectable marker which can be used tomonitor integration. Typically, several kilobases of unaltered flankingDNA (both at the 5′ and 3′ ends) are included in the vector [see, e.g.,Thomas and Capecchi, Cell, 51:503 (1987) for a description of homologousrecombination vectors]. The vector is introduced into an embryonic stemcell line (e.g., by electroporation) and cells in which the introducedDNA has homologously recombined with the endogenous DNA are selected[see, e.g., Li et al., Cell, 69:915 (1992)]. The selected cells are theninjected into a blastocyst of an animal (e.g., a mouse OF rat) to formaggregation chimeras [see, e.g., Bradley, in Teratocarcinomas andEmbryonic Stem Cells: A Practical Approach, E. J. Robertson, ed. (IRL,Oxford, 1987), pp. 113-152]. A chimeric embryo can then be implantedinto a suitable pseudopregnant female foster animal and the embryobrought to term to create a “knock out” animal. Progeny harboring thehomologously recombined DNA in their germ cells can be identified bystandard techniques and used to breed animals in which all cells of theanimal contain the homologously recombined DNA. Knockout animals can becharacterized for instance, by their ability to defend against certainpathological conditions and by their development of pathologicalconditions due to absence of the PRO10282 polypeptide.

The efficacy of antibodies specifically binding the polypeptidesidentified herein and other drug candidates, can be tested also in thetreatment of spontaneous animal tumors. A suitable target for suchstudies is the feline oral squamous cell carcinoma (SCC). Feline oralSCC is a highly invasive, malignant tumor that is the most common oralmalignancy of cats, accounting for over 60% of the oral tumors reportedin this species. It rarely metastasizes to distant sites, although thislow incidence of metastasis may merely be a reflection of the shortsurvival times for cats with this tumor. These tumors are usually notamenable to surgery, primarily because of the anatomy of the feline oralcavity. At present, there is no effective treatment for this tumor.Prior to entry into the study, each cat undergoes complete clinicalexamination, biopsy, and is scanned by computed tomography (CT). Catsdiagnosed with sublingual oral squamous cell tumors are excluded fromthe study. The tongue can become paralyzed as a result of such tumor,and even if the treatment kills the tumor, the animals may not be ableto feed themselves. Each cat is treated repeatedly, over a longer periodof time. Photographs of the tumors will be taken daily during thetreatment period, and at each subsequent recheck. After treatment, eachcat undergoes another CT scan. CT scans and thoracic radiograms areevaluated every 8 weeks thereafter. The data are evaluated fordifferences in survival, response and toxicity as compared to controlgroups. Positive response may require evidence of tumor regression,preferably with improvement of quality of life and/or increased lifespan.

In addition, other spontaneous animal tumors, such as fibrosarcoma,adenocarcinoma, lymphoma, chrondroma, leiomyosarcoma of dogs, cats, andbaboons can also be tested. Of these mammary adenocarcinoma in dogs andcats is a preferred model as its appearance and behavior are verysimilar to those in humans. However, the use of this model is limited bythe rare occurrence of this type of tumor in animals.

K. Screening Assays for Drug Candidates

Screening assays for drug candidates are designed to identify compoundsthat bind or complex with the polypeptides encoded by the genesidentified herein, or otherwise interfere with the interaction of theencoded polypeptides with other cellular proteins. Such screening assayswill include assays amenable to high-throughput screening of chemicallibraries, making them particularly suitable for identifying smallmolecule drug candidates. Small molecules contemplated include syntheticorganic or inorganic compounds, including peptides, preferably solublepeptides, (poly)peptide-immunoglobulin fusions, and, in particular,antibodies including, without limitation, poly- and monoclonalantibodies and antibody fragments, single-chain antibodies,anti-idiotypic antibodies, and chimeric or humanized versions of suchantibodies or fragments, as well as human antibodies and antibodyfragments. The assays can be performed in a variety of formats,including protein-protein binding assays, biochemical screening assays,immunoassays and cell based assays, which are well characterized in theart.

All assays are common in that they call for contacting the drugcandidate with a polypeptide encoded by a nucleic acid identified hereinunder conditions and for a time sufficient to allow these two componentsto interact.

In binding assays, the interaction is binding and the complex formed canbe isolated or detected in the reaction mixture. In a particularembodiment, the polypeptide encoded by the gene identified herein or thedrug candidate is immobilized on a solid phase, e.g., on a microtiterplate, by covalent or non-covalent attachments. Non-covalent attachmentgenerally is accomplished by coating the solid surface with a solutionof the polypeptide and drying. Alternatively, an immobilized antibody,e.g., a monoclonal antibody, specific for the polypeptide to beimmobilized can be used to anchor it to a solid surface. The assay isperformed by adding the non-immobilized component, which may be labeledby a detectable label, to the immobilized component, e.g., the coatedsurface containing the anchored component. When the reaction iscomplete, the non-reacted components are removed, e.g., by washing, andcomplexes anchored on the solid surface are detected. When theoriginally non-immobilized component carries a detectable label, thedetection of label immobilized on the surface indicates that complexingoccurred. Where the originally non-immobilized component does not carrya label, complexing can be detected, for example, by using a labeledantibody specifically binding the immobilized complex.

If the candidate compound interacts with but does not bind to aparticular PRO10282 polypeptide encoded by a gene identified herein, itsinteraction with that polypeptide can be assayed by methods well knownfor detecting protein-protein interactions. Such assays includetraditional approaches, such as, cross-linking, co-immunoprecipitation,and co-purification through gradients or chromatographic columns. Inaddition, protein-protein interactions can be monitored by using ayeast-based genetic system described by Fields and co-workers [Fieldsand Song, Nature, 340:245-246 (1989); Chien et al., Proc. Natl. Acad.Sci. USA, 88: 9578-9582 (1991)] as disclosed by Chevray and Nathans.Proc. Natl. Acad. Sci. USA, 89:5789-5793 (1991)]. Many transcriptionalactivators, such as yeast GAL4, consist of two physically discretemodular domains, one acting as the DNA-binding domain, while the otherone functioning as the transcription activation domain. The yeastexpression system described in the foregoing publications (generallyreferred to as the “two-hybrid system”) takes advantage of thisproperty, and employs two hybrid proteins, one in which the targetprotein is fused to the DNA-binding domain of GAL4, and another, inwhich candidate activating proteins are fused to the activation domain.The expression of a GAL1-lacZ reporter gene under control of aGAL4-activated promoter depends on reconstitution of GAL4 activity viaprotein-protein interaction. Colonies containing interactingpolypeptides are detected with a chromogenic substrate for-galactosidase. A complete kit (MATCHMAKER™) for identifyingprotein-protein interactions between two specific proteins using thetwo-hybrid technique is commercially available from Clontech. Thissystem can also be extended to map protein domains involved in specificprotein interactions as well as to pinpoint amino acid residues that arecrucial for these interactions.

Compounds that interfere with the interaction of a PRO10282-encodinggene identified herein and other intra- or extracellular components canbe tested as follows: usually a reaction mixture is prepared containingthe product of the amplified gene and the intra- or extracellularcomponent under conditions and for a time allowing for the interactionand binding of the two products. To test the ability of a test compoundto inhibit binding, the reaction is run in the absence and in thepresence of the test compound. In addition, a placebo may be added to athird reaction mixture, to serve as positive control. The binding(complex formation) between the test compound and the intra- orextracellular component present in the mixture is monitored as describedhereinabove. The formation of a complex in the control reaction(s) butnot in the reaction mixture containing the test compound indicates thatthe test compound interferes with the interaction of the test compoundand its reaction partner.

To assay for antagonists, the PRO10282 polypeptide may be added to acell along with the compound to be screened for a particular activityand the ability of the compound to inhibit the activity of interest inthe presence of the PRO10282 polypeptide indicates that the compound isan antagonist to the PRO10282 polypeptide. Alternatively, antagonistsmay be detected by combining the PRO10282 polypeptide and a potentialantagonist with membrane-bound PRO10282 polypeptide receptors orrecombinant receptors under appropriate conditions for a competitiveinhibition assay. The PRO10282 polypeptide can be labeled, such as byradioactivity, such that the number of PRO10282 polypeptide moleculesbound to the receptor can be used to determine the effectiveness of thepotential antagonist. The gene encoding the receptor can be identifiedby numerous methods known to those of skill in the art, for example,ligand panning and FACS sorting. Coligan et al., Current Protocols inImmun., 1(2): Chapter 5 (1991). Preferably, expression cloning isemployed wherein polyadenylated RNA is prepared from a cell responsiveto the PRO10282 polypeptide and a cDNA library created from this RNA isdivided into pools and used to transfect COS cells or other cells thatare not responsive to the PRO10282 polypeptide. Transfected cells thatare grown on glass slides are exposed to labeled PRO10282 polypeptide.The PRO10282 polypeptide can be labeled by a variety of means includingiodination or inclusion of a recognition site for a site-specificprotein kinase. Following fixation and incubation, the slides aresubjected to autoradiographic analysis. Positive pools are identifiedand sub-pools are prepared and re-transfected using an interactivesub-pooling and re-screening process, eventually yielding a single clonethat encodes the putative receptor.

As an alternative approach for receptor identification, labeled PRO10282polypeptide can be photoaffinity-linked with cell membrane or extractpreparations that express the receptor molecule. Cross-linked materialis resolved by PAGE and exposed to X-ray film. The labeled complexcontaining the receptor can be excised, resolved into peptide fragments,and subjected to protein micro-sequencing. The amino acid sequenceobtained from micro-sequencing would be used to design a set ofdegenerate oligonucleotide probes to screen a cDNA library to identifythe gene encoding the putative receptor.

In another assay for antagonists, mammalian cells or a membranepreparation expressing the receptor would be incubated with labeledPRO10282 polypeptide in the presence of the candidate compound. Theability of the compound to enhance or block this interaction could thenbe measured.

More specific examples of potential antagonists include anoligonucleotide that binds to the fusions of immunoglobulin with thePRO10282 polypeptide, and, in particular, antibodies including, withoutlimitation, poly- and monoclonal antibodies and antibody fragments,single-chain antibodies, anti-idiotypic antibodies, and chimeric orhumanized versions of such antibodies or fragments, as well as humanantibodies and antibody fragments. Alternatively, a potential antagonistmay be a closely related protein, for example, a mutated form of thePRO10282 polypeptide that recognizes the receptor but imparts no effect,thereby competitively inhibiting the action of the PRO10282 polypeptide.

Another potential PRO10282 polypeptide antagonist is an antisense RNA orDNA construct prepared using antisense technology, where, e.g., anantisense RNA or DNA molecule acts to block directly the translation ofmRNA by hybridizing to targeted mRNA and preventing protein translation.Antisense technology can be used to control gene expression throughtriple-helix formation or antisense DNA or RNA, both of which methodsare based on binding of a polynucleotide to DNA or RNA. For example, the5′ coding portion of the polynucleotide sequence, which encodes themature PRO10282 polypeptide herein, is used to design an antisense RNAoligonucleotide of from about 10 to 40 base pairs in length. A DNAoligonucleotide is designed to be complementary to a region of the geneinvolved in transcription (triple helix—see, Lee et al., Nucl. AcidsRes., 6:3073 (1979); Cooney et al., Science, 241: 456 (1988); Dervan etal., Science, 251:1360 (1991)), thereby preventing transcription and theproduction of the PRO10282 polypeptide. The antisense RNAoligonucleotide hybridizes to the mRNA in vivo and blocks translation ofthe mRNA molecule into the PRO10282 polypeptide (antisense—Okano,Neurochem., 56:560 (1991); Oligodeoxynucleotides as Antisense Inhibitorsof Gene Expression (CRC Press: Boca Raton, Fla., 1988). Theoligonucleotides described above can also be delivered to cells suchthat the antisense RNA or DNA may be expressed in vivo to inhibitproduction of the PRO10282 polypeptide. When antisense DNA is used,oligodeoxyribonucleotides derived from the translation-initiation site,e.g., between about −10 and +10 positions of the target gene nucleotidesequence, are preferred.

Antisense RNA or DNA molecules are generally at least about 5 bases inlength, about 10 bases in length, about 15 bases in length, about 20bases in length, about 25 bases in length, about 30 bases in length,about 35 bases in length, about 40 bases in length, about 45 bases inlength, about 50 bases in length, about 55 bases in length, about 60bases in length, about 65 bases in length, about 70 bases in length,about 75 bases in length, about 80 bases in length, about 85 bases inlength, about 90 bases in length, about 95 bases in length, about 100bases in length, or more.

Potential antagonists include small molecules that bind to the activesite, the receptor binding site, or growth factor or other relevantbinding site of the PRO10282 polypeptide, thereby blocking the normalbiological activity of the PRO10282 polypeptide. Examples of smallmolecules include, but are not limited to, small peptides orpeptide-like molecules, preferably soluble peptides, and syntheticnon-peptidyl organic or inorganic compounds.

Ribozymes are enzymatic RNA molecules capable of catalyzing the specificcleavage of RNA. Ribozymes act by sequence-specific hybridization to thecomplementary target RNA, followed by endonucleolytic cleavage. Specificribozyme cleavage sites within a potential RNA target can be identifiedby known techniques. For further details see, e.g., Rossi, CurrentBiology, 4:469-471 (1994), and PCT publication No. WO 97/33551(published Sep. 18, 1997).

Nucleic acid molecules in triple-helix formation used to inhibittranscription should be single-stranded and composed ofdeoxynucleotides. The base composition of these oligonucleotides isdesigned such that it promotes triple-helix formation via Hoogsteenbase-pairing rules, which generally require sizeable stretches ofpurines or pyrimidines on one strand of a duplex. For further detailssee, e.g., PCT publication No. WO 97/33551, supra.

These small molecules can be identified by any one or more of thescreening assays discussed hereinabove and/or by any other screeningtechniques well known for those skilled in the art.

L. Uses for PRO10282 (Stra6)

Nucleotide sequences (or their complement) encoding PRO10282 (Stra6)polypeptides have various applications in the art of molecular biology,including uses as hybridization probes, in chromosome and gene mappingand in the generation of anti-sense RNA and DNA. PRO10282 nucleic acidwill also be useful for the preparation of PRO10282 polypeptides by therecombinant techniques described herein.

The full-length native sequence PRO10282 gene (SEQ ID NO:1), or PRO19578gene (SEQ ID NO: 4), or portions thereof, may be used as hybridizationprobes for a cDNA library to isolate the full-length PRO10282 orPRO19578 cDNA or to isolate still other cDNAs (for instance, thoseencoding further naturally-occurring variants of PRO10282, or PRO10282from other species) which have a desired sequence identity to thePRO10282 coding sequence disclosed in FIG. 1 (SEQ ID NO:1) or thePRO19578 coding sequence disclosed in FIG. 6 (SEQ ID NO: 4). Optionally,the length of the probes will be about 20 to about 50 bases. Thehybridization probes may be derived from at least partially novelregions of the nucleotide sequence of SEQ ID NO:1 wherein those regionsmay be determined without undue experimentation or from genomicsequences including promoters, enhancer elements and introns of thenative PRO10282 or PRO19578 gene. By way of example, a screening methodwill comprise isolating the coding region of the PRO10282 or PRO19578gene using the known DNA sequence to synthesize a selected probe ofabout 40 bases. Hybridization probes may be labeled by a variety oflabels, including radionucleotides such as ³²P or ³⁵S, or enzymaticlabels such as alkaline phosphatase coupled to the probe viaavidin/biotin coupling systems. Labeled probes having a sequencecomplementary to that of the PRO10282 or PRO19578 gene of the presentinvention can be used to screen libraries of human cDNA, genomic DNA ormRNA to determine which members of such libraries the probe hybridizesto. Hybridization techniques are described in further detail in theExamples below.

Any EST sequences disclosed in the present application may similarly beemployed as probes, using the methods disclosed herein.

Other useful fragments of the PRO10282 (Stra6) nucleic acids includeantisense or sense oligonucleotides comprising a singe-stranded nucleicacid sequence (either RNA or DNA) capable of binding to target PRO10282(Stra6) mRNA (sense) or PRO10282 (Stra6) DNA (antisense) sequences.Antisense or sense oligonucleotides, according to the present invention,comprise a fragment of the coding region of PRO10282 (Stra6) DNA. Such afragment generally comprises at least about 14 nucleotides, preferablyfrom about 14 to 30 nucleotides. The ability to derive an antisense or asense oligonucleotide, based upon a cDNA sequence encoding a givenprotein is described in, for example, Stein and Cohen (Cancer Res.48:2659, 1988) and van der Krol et al. (BioTechniques 6:958, 1988).

Binding of antisense or sense oligonucleotides to target nucleic acidsequences results in the formation of duplexes that block transcriptionor translation of the target sequence by one of several means, includingenhanced degradation of the duplexes, premature termination oftranscription or translation, or by other means. The antisenseoligonucleotides thus may be used to block expression of PRO10282(Stra6) proteins. Antisense or sense oligonucleotides further compriseoligonucleotides having modified sugar-phosphodiester backbones (orother sugar linkages, such as those described in WO 91/06629) andwherein such sugar linkages are resistant to endogenous nucleases. Sucholigonucleotides with resistant sugar linkages are stable in vivo (i.e.,capable of resisting enzymatic degradation) but retain sequencespecificity to be able to bind to target nucleotide sequences.

Other examples of sense or antisense oligonucleotides include thoseoligonucleotides which are covalently linked to organic moieties, suchas those described in WO 90/10048, and other moieties that increasesaffinity of the oligonucleotide for a target nucleic acid sequence, suchas poly(L-lysine). Further still, intercalating agents, such asellipticine, and alkylating agents or metal complexes may be attached tosense or antisense oligonucleotides to modify binding specificities ofthe antisense or sense oligonucleotide for the target nucleotidesequence.

Antisense or sense oligonucleotides may be introduced into a cellcontaining the target nucleic acid sequence by any gene transfer method,including, for example, CaPO₄-mediated DNA transfection,electroporation, or by using gene transfer vectors such as Epstein-Barrvirus. In a preferred procedure, an antisense or sense oligonucleotideis inserted into a suitable retroviral vector. A cell containing thetarget nucleic acid sequence is contacted with the recombinantretroviral vector, either in vivo or ex vivo. Suitable retroviralvectors include, but are not limited to, those derived from the murineretrovirus M-MuLV, N2 (a retrovirus derived from M-MuLV), or the doublecopy vectors designated DCT5A, DCT5B and DCT5C (see WO 90/13641).

Sense or antisense oligonucleotides also may be introduced into a cellcontaining the target nucleotide sequence by formation of a conjugatewith a ligand-binding molecule, as described in WO 91/04753. Suitableligand binding molecules include, but are not limited to, cell surfacereceptors, growth factors, other cytokines, or other ligands that bindto cell surface receptors. Preferably, conjugation of the ligand-bindingmolecule does not substantially interfere with the ability of theligand-binding molecule to bind to its corresponding molecule orreceptor, or block entry of the sense or antisense oligonucleotide orits conjugated version into the cell.

Alternatively, a sense or an antisense oligonucleotide may be introducedinto a cell containing the target nucleic acid sequence by formation ofan oligonucleotide-lipid complex, as described in WO 90/10448. The senseor antisense oligonucleotide-lipid complex is preferably dissociatedwithin the cell by an endogenous lipase.

The probes may also be employed in PCR techniques to generate a pool ofsequences for identification of closely related PRO10282 codingsequences.

Nucleotide sequences encoding a native sequence PRO10282 (Stra6)polypeptide can also be used to construct hybridization probes formapping the gene, which encodes that native sequence PRO10282 (Stra6)and for the genetic analysis of individuals with genetic disorders. Thenucleotide sequences provided herein may be mapped to a chromosome andspecific regions of a chromosome using known techniques, such as in situhybridization, linkage analysis against known chromosomal markers, andhybridization screening with libraries.

When the coding sequences for PRO10282 encode a protein which binds toanother protein (for example, where the PRO10282 is a receptor), thePRO10282 can be used in assays to identify the other proteins ormolecules involved in the binding interaction. By such methods,inhibitors of the receptor/ligand binding interaction can be identified.Proteins involved in such binding interactions can also be used toscreen for peptide or small molecule inhibitors or agonists of thebinding interaction. Also, the receptor PRO10282 (Stra6) can be used toisolate correlative ligand(s). Screening assays can be designed to findlead compounds that mimic the biological activity of a native PRO10282(Stra6) or native polypeptide binding to PRO10282 (“PRO10282 bindingprotein”). Such screening assays will include assays amenable tohigh-throughput screening of chemical libraries, making themparticularly suitable for identifying small molecule drug candidates.Small molecules contemplated include synthetic organic or inorganiccompounds. The assays can be performed in a variety of formats,including protein-protein binding assays, biochemical screening assays,immunoassays and cell based assays, which are well characterized in theart.

Nucleic acids which encode PRO10282 or its modified forms can also beused to generate either transgenic animals or “knock out” animals which,in turn, are useful in the development and screening of therapeuticallyuseful reagents. A transgenic animal (e.g., a mouse or rat) is an animalhaving cells that contain a transgene, which transgene was introducedinto the animal or an ancestor of the animal at a prenatal, e.g., anembryonic stage. A transgene is a DNA which is integrated into thegenome of a cell from which a transgenic animal develops. In oneembodiment, cDNA encoding PRO10282 can be used to clone genomic DNAencoding PRO10282 in accordance with established techniques and thegenomic sequences used to generate transgenic animals that contain cellswhich express DNA encoding PRO10282. Methods for generating transgenicanimals, particularly animals such as mice or rats, have becomeconventional in the art and are described, for example, in U.S. Pat.Nos. 4,736,866 and 4,870,009. Typically, particular cells would betargeted for PRO10282 transgene incorporation with tissue-specificenhancers. Transgenic animals that include a copy of a transgeneencoding PRO10282 introduced into the germ line of the animal at anembryonic stage can be used to examine the effect of increasedexpression of DNA encoding PRO10282. Such animals can be used as testeranimals for reagents thought to confer protection from, for example,pathological conditions associated with its overexpression. Inaccordance with this facet of the invention, an animal is treated withthe reagent and a reduced incidence of the pathological condition,compared to untreated animals bearing the transgene, would indicate apotential therapeutic intervention for the pathological condition.

Alternatively, non-human homologues of PRO10282 can be used to constructa PRO10282 “knock out” animal which has a defective or altered geneencoding PRO10282 as a result of homologous recombination between theendogenous gene encoding PRO10282 and altered genomic DNA encodingPRO10282 introduced into an embryonic stem cell of the animal. Forexample, cDNA encoding PRO10282 can be used to clone genomic DNAencoding PRO10282 in accordance with established techniques. A portionof the genomic DNA encoding PRO10282 can be deleted or replaced withanother gene, such as a gene encoding a selectable marker which can beused to monitor integration. Typically, several kilobases of unalteredflanking DNA (both at the 5′ and 3′ ends) are included in the vector[see e.g., Thomas and Capecchi, Cell, 51:503 (1987) for a description ofhomologous recombination vectors]. The vector is introduced into anembryonic stem cell line (e.g., by electroporation) and cells in whichthe introduced DNA has homologously recombined with the endogenous DNAare selected [see e.g., Li et al., Cell, 69:915 (1992)]. The selectedcells are then injected into a blastocyst of an animal (e.g., a mouse orrat) to form aggregation chimeras [see e.g., Bradley, inTeratocarcinomas and Embryonic Stem Cells: A Practical Approach, E. J.Robertson, ed. (IRL, Oxford, 1987), pp. 113-152]. A chimeric embryo canthen be implanted into a suitable pseudopregnant female foster animaland the embryo brought to term to create a “knock out” animal. Progenyharboring the homologously recombined DNA in their germ cells can beidentified by standard techniques and used to breed animals in which allcells of the animal contain the homologously recombined DNA. Knockoutanimals can be characterized for instance, for their ability to defendagainst certain pathological conditions and for their development ofpathological conditions due to absence of the PRO10282 polypeptide.

Nucleic acid encoding the PRO10282 polypeptides may also be used in genetherapy. In gene therapy applications, genes are introduced into cellsin order to achieve in vivo synthesis of a therapeutically effectivegenetic product, for example for replacement of a defective gene. “Genetherapy” includes both conventional gene therapy where a lasting effectis achieved by a single treatment, and the administration of genetherapeutic agents, which involves the one time or repeatedadministration of a therapeutically effective DNA or mRNA. AntisenseRNAs and DNAs can be used as therapeutic agents for blocking theexpression of certain genes in vivo. It has already been shown thatshort antisense oligonucleotides can be imported into cells where theyact as inhibitors, despite their low intracellular concentrations causedby their restricted uptake by the cell membrane. (Zamecnik et al., Proc.Natl. Acad. Sci. USA 83:4143-4146 [1986]). The oligonucleotides can bemodified to enhance their uptake, e.g. by substituting their negativelycharged phosphodiester groups by uncharged groups.

There are a variety of techniques available for introducing nucleicacids into viable cells. The techniques vary depending upon whether thenucleic acid is transferred into cultured cells in vitro, or in vivo inthe cells of the intended host. Techniques suitable for the transfer ofnucleic acid into mammalian cells in vitro include the use of liposomes,electroporation, microinjection, cell fusion, DEAE-dextran, the calciumphosphate precipitation method, etc. The currently preferred in vivogene transfer techniques include transfection with viral (typicallyretroviral) vectors and viral coat protein-liposome mediatedtransfection (Dzau et al., Trends in Biotechnology 11, 205-210 [1993]).In some situations it is desirable to provide the nucleic acid sourcewith an agent that targets the target cells, such as an antibodyspecific for a cell surface membrane protein or the target cell, aligand for a receptor on the target cell, etc. Where liposomes areemployed, proteins which bind to a cell surface membrane proteinassociated with endocytosis may be used for targeting and/or tofacilitate uptake, e.g. capsid proteins or fragments thereof tropic fora particular cell type, antibodies for proteins which undergointernalization in cycling, proteins that target intracellularlocalization and enhance intracellular half-life. The technique ofreceptor-mediated endocytosis is described, for example, by Wu et al.,J. Biol. Chem. 262, 4429-4432 (1987); and Wagner et al., Proc. Natl.Acad. Sci. USA 87, 3410-3414 (1990). For review of gene marking and genetherapy protocols see Anderson et al., Science 256, 808-813 (1992).

The PRO10282 polypeptides described herein may also be employed asmolecular weight markers for protein electrophoresis purposes.

The nucleic acid molecules encoding the PRO10282 polypeptides orfragments thereof described herein are useful for chromosomeidentification. In this regard, there exists an ongoing need to identifynew chromosome markers, since relatively few chromosome markingreagents, based upon actual sequence data are presently available. EachPRO10282 nucleic acid molecule of the present invention can be used as achromosome marker.

The PRO10282 polypeptides and nucleic acid molecules of the presentinvention may also be used for tissue typing, wherein the PRO10282polypeptides of the present invention may be differentially expressed inone tissue as compared to another. PRO10282 nucleic acid molecules willfind use for generating probes for PCR, Northern analysis, Southernanalysis and Western analysis.

The PRO10282 polypeptides described herein may also be employed astherapeutic agents. The PRO10282 polypeptides of the present inventioncan be formulated according to known methods to prepare pharmaceuticallyuseful compositions, whereby the PRO10282 product hereof is combined inadmixture with a pharmaceutically acceptable carrier vehicle.Therapeutic formulations are prepared for storage by mixing the activeingredient having the desired degree of purity with optionalphysiologically acceptable carriers, excipients or stabilizers(Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)),in the form of lyophilized formulations or aqueous solutions. Acceptablecarriers, excipients or stabilizers are nontoxic to recipients at thedosages and concentrations employed, and include buffers such asphosphate, citrate and other organic acids; antioxidants includingascorbic acid; low molecular weight (less than about 10 residues)polypeptides; proteins, such as serum albumin, gelatin orimmunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone,amino acids such as glycine, glutamine, asparagine, arginine or lysine;monosaccharides, disaccharides and other carbohydrates includingglucose, mannose, or dextrins; chelating agents such as EDTA; sugaralcohols such as mannitol or sorbitol; salt-forming counterions such assodium; and/or nonionic surfactants such as TWEEN™, PLURONICS™ or PEG.

The formulations to be used for in vivo administration must be sterile.This is readily accomplished by filtration through sterile filtrationmembranes, prior to or following lyophilization and reconstitution.

Therapeutic compositions herein generally are placed into a containerhaving a sterile access port, for example, an intravenous solution bagor vial having a stopper pierceable by a hypodermic injection needle.

The route of administration is in accord with known methods, e.g.injection or infusion by intravenous, intraperitoneal, intracerebral,intramuscular, intraocular, intraarterial or intralesional routes,topical administration, or by sustained release systems.

Dosages and desired drug concentrations of pharmaceutical compositionsof the present invention may vary depending on the particular useenvisioned. The determination of the appropriate dosage or route ofadministration is well within the skill of an ordinary physician. Animalexperiments provide reliable guidance for the determination of effectivedoses for human therapy. Interspecies scaling of effective doses can beperformed following the principles laid down by Mordenti, J. andChappell, W. “The use of interspecies scaling in toxicokinetics” InToxicokinetics and New Drug Development, Yacobi et al., Eds., PergamonPress, New York 1989, pp. 42-96.

When in vivo administration of a PRO10282 polypeptide or agonist orantagonist thereof is employed, normal dosage amounts may vary fromabout 10 ng/kg to up to 100 mg/kg of mammal body weight or more per day,preferably about 1 g/kg/day to 10 mg/kg/day, depending upon the route ofadministration. Guidance as to particular dosages and methods ofdelivery is provided in the literature; see, for example, U.S. Pat. No.4,657,760; 5,206,344; or 5,225,212. It is anticipated that differentformulations will be effective for different treatment compounds anddifferent disorders, that administration targeting one organ or tissue,for example, may necessitate delivery in a manner different from that toanother organ or tissue.

Where sustained-release administration of a PRO10282 polypeptide isdesired in a formulation with release characteristics suitable for thetreatment of any disease or disorder requiring administration of thePRO10282 polypeptide, microencapsulation of the PRO10282 polypeptide iscontemplated. Microencapsulation of recombinant proteins for sustainedrelease has been successfully performed with human growth hormone(rhGH), interferon-(rhIFN-), interleukin-2, and MN rgp120. Johnson etal., Nat. Med., 2:795-799 (1996); Yasuda, Biomed. Ther., 27:1221-1223(1993); Hora et al., Bio/Technology, 8:755-758 (1990); Cleland, “Designand Production of Single Immunization Vaccines Using PolylactidePolyglycolide Microsphere Systems,” in Vaccine Design: The Subunit andAdjuvant Approach, Powell and Newman, eds, (Plenum Press: New York,1995), pp. 439-462; WO 97/03692, WO 96/40072, WO 96/07399; and U.S. Pat.No. 5,654,010.

The sustained-release formulations of these proteins were developedusing poly-lactic-coglycolic acid (PLGA) polymer due to itsbiocompatibility and wide range of biodegradable properties. Thedegradation products of PLGA, lactic and glycolic acids, can be clearedquickly within the human body. Moreover, the degradability of thispolymer can be adjusted from months to years depending on its molecularweight and composition. Lewis, “Controlled release of bioactive agentsfrom lactide/glycolide polymer,” in: M. Chasin and R. Langer (Eds.),Biodegradable Polymers as Drug Delivery Systems (Marcel Dekker: NewYork, 1990), pp. 1-41.

This invention encompasses methods of screening compounds to identifythose that mimic the PRO10282 polypeptide (agonists) or prevent theeffect of the PRO10282 polypeptide (antagonists). Screening assays forantagonist drug candidates are designed to identify compounds that bindor complex with the PRO10282 polypeptides encoded by the genesidentified herein, or otherwise interfere with the interaction of theencoded polypeptides with other cellular proteins. Such screening assayswill include assays amenable to high-throughput screening of chemicallibraries, making them particularly suitable for identifying smallmolecule drug candidates.

The assays can be performed in a variety of formats, includingprotein-protein binding assays, biochemical screening assays,immunoassays, and cell-based assays, which are well characterized in theart.

All assays for antagonists are common in that they call for contactingthe drug candidate with a PRO10282 polypeptide encoded by a nucleic acididentified herein under conditions and for a time sufficient to allowthese two components to interact.

In binding assays, the interaction is binding and the complex formed canbe isolated or detected in the reaction mixture. In a particularembodiment, the PRO10282 polypeptide encoded by the gene identifiedherein or the drug candidate is immobilized on a solid phase, e.g., on amicrotiter plate, by covalent or non-covalent attachments. Non-covalentattachment generally is accomplished by coating the solid surface with asolution of the PRO10282 polypeptide and drying. Alternatively, animmobilized antibody, e.g., a monoclonal antibody, specific for thePRO10282 polypeptide to be immobilized can be used to anchor it to asolid surface. The assay is performed by adding the non-immobilizedcomponent, which may be labeled by a detectable label, to theimmobilized component, e.g., the coated surface containing the anchoredcomponent. When the reaction is complete, the non-reacted components areremoved, e.g., by washing, and complexes anchored on the solid surfaceare detected. When the originally non-immobilized component carries adetectable label, the detection of label immobilized on the surfaceindicates that complexing occurred. Where the originally non-immobilizedcomponent does not carry a label, complexing can be detected, forexample, by using a labeled antibody specifically binding theimmobilized complex.

If the candidate compound interacts with but does not bind to aparticular PRO10282 polypeptide encoded by a gene identified herein, itsinteraction with that polypeptide can be assayed by methods well knownfor detecting protein-protein interactions. Such assays includetraditional approaches, such as, e.g., cross-linking,co-immunoprecipitation, and co-purification through gradients orchromatographic columns. In addition, protein-protein interactions canbe monitored by using a yeast-based genetic system described by Fieldsand co-workers (Fields and Song, Nature (London), 340:245-246 (1989);Chien et al., Proc. Natl. Acad. Sci. USA, 88:9578-9582 (1991)) asdisclosed by Chevray and Nathans, Proc. Natl. Acad. Sci. USA, 89:5789-5793 (1991). Many transcriptional activators, such as yeast GAL4,consist of two physically discrete modular domains, one acting as theDNA-binding domain, the other one functioning as thetranscription-activation domain. The yeast expression system describedin the foregoing publications (generally referred to as the “two-hybridsystem”) takes advantage of this property, and employs two hybridproteins, one in which the target protein is fused to the DNA-bindingdomain of GAL4, and another, in which candidate activating proteins arefused to the activation domain. The expression of a GAL1-lacZ reportergene under control of a GAL4-activated promoter depends onreconstitution of GAL4 activity via protein-protein interaction.Colonies containing interacting polypeptides are detected with achromogenic substrate for -galactosidase. A complete kit (MATCHMAKER™)for identifying protein-protein interactions between two specificproteins using the two-hybrid technique is commercially available fromClontech. This system can also be extended to map protein domainsinvolved in specific protein interactions as well as to pinpoint aminoacid residues that are crucial for these interactions.

Compounds that interfere with the interaction of a gene encoding aPRO10282 polypeptide identified herein and other intra- or extracellularcomponents can be tested as follows: usually a reaction mixture isprepared containing the product of the gene and the intra- orextracellular component under conditions and for a time allowing for theinteraction and binding of the two products. To test the ability of acandidate compound to inhibit binding, the reaction is run in theabsence and in the presence of the test compound. In addition, a placebomay be added to a third reaction mixture, to serve as positive control.The binding (complex formation) between the test compound and the intra-or extracellular component present in the mixture is monitored asdescribed hereinabove. The formation of a complex in the controlreaction(s) but not in the reaction mixture containing the test compoundindicates that the test compound interferes with the interaction of thetest compound and its reaction partner.

To assay for antagonists, the PRO10282 polypeptide may be added to acell along with the compound to be screened for a particular activityand the ability of the compound to inhibit the activity of interest inthe presence of the PRO10282 polypeptide indicates that the compound isan antagonist to the PRO10282 polypeptide. Alternatively, antagonistsmay be detected by combining the PRO10282 polypeptide and a potentialantagonist with membrane-bound PRO10282 polypeptide receptors orrecombinant receptors under appropriate conditions for a competitiveinhibition assay. The PRO10282 polypeptide can be labeled, such as byradioactivity, such that the number of PRO10282 polypeptide moleculesbound to the receptor can be used to determine the effectiveness of thepotential antagonist. The gene encoding the receptor can be identifiedby numerous methods known to those of skill in the art, for example,ligand panning and FACS sorting. Coligan et al., Current Protocols inImmun., 1(2): Chapter 5 (1991). Preferably, expression cloning isemployed wherein polyadenylated RNA is prepared from a cell responsiveto the PRO10282 polypeptide and a cDNA library created from this RNA isdivided into pools and used to transfect COS cells or other cells thatare not responsive to the PRO10282 polypeptide. Transfected cells thatare grown on glass slides are exposed to labeled PRO10282 polypeptide.The PRO10282 polypeptide can be labeled by a variety of means includingiodination or inclusion of a recognition site for a site-specificprotein kinase. Following fixation and incubation, the slides aresubjected to autoradiographic analysis. Positive pools are identifiedand sub-pools are prepared and re-transfected using an interactivesub-pooling and re-screening process, eventually yielding a single clonethat encodes the putative receptor.

As an alternative approach for receptor identification, labeled PRO10282polypeptide can be photoaffinity-linked with cell membrane or extractpreparations that express the receptor molecule. Cross-linked materialis resolved by PAGE and exposed to X-ray film. The labeled complexcontaining the receptor can be excised, resolved into peptide fragments,and subjected to protein micro-sequencing. The amino acid sequenceobtained from micro-sequencing would be used to design a set ofdegenerate oligonucleotide probes to screen a cDNA library to identifythe gene encoding the putative receptor.

In another assay for antagonists, mammalian cells or a membranepreparation expressing the receptor would be incubated with labeledPRO10282 polypeptide in the presence of the candidate compound. Theability of the compound to enhance or block this interaction could thenbe measured.

More specific examples of potential antagonists include anoligonucleotide that binds to the fusions of immunoglobulin withPRO10282 polypeptide, and, in particular, antibodies including, withoutlimitation, poly- and monoclonal antibodies and antibody fragments,single-chain antibodies, anti-idiotypic antibodies, and chimeric orhumanized versions of such antibodies or fragments, as well as humanantibodies and antibody fragments. Alternatively, a potential antagonistmay be a closely related protein, for example, a mutated form of thePRO10282 polypeptide that recognizes the receptor but imparts no effect,thereby competitively inhibiting the action of the PRO10282 polypeptide.

Another potential PRO10282 polypeptide antagonist is an antisense RNA orDNA construct prepared using antisense technology, where, e.g., anantisense RNA or DNA molecule acts to block directly the translation ofmRNA by hybridizing to targeted mRNA and preventing protein translation.Antisense technology can be used to control gene expression throughtriple-helix formation or antisense DNA or RNA, both of which methodsare based on binding of a polynucleotide to DNA or RNA. For example, the5′ coding portion of the polynucleotide sequence, which encodes themature PRO10282 polypeptides herein, is used to design an antisense RNAoligonucleotide of from about 10 to 40 base pairs in length. A DNAoligonucleotide is designed to be complementary to a region of the geneinvolved in transcription (triple helix—see Lee et al., Nucl. AcidsRes., 6:3073 (1979); Cooney et al., Science, 241: 456 (1988); Dervan etal., Science, 251:1360 (1991)), thereby preventing transcription and theproduction of the PRO10282 polypeptide. The antisense RNAoligonucleotide hybridizes to the mRNA in vivo and blocks translation ofthe mRNA molecule into the PRO10282 polypeptide (antisense—Okano,Neurochem., 56:560 (1991); Oligodeoxynucleotides as Antisense Inhibitorsof Gene Expression (CRC Press: Boca Raton, Fla., 1988). Theoligonucleotides described above can also be delivered to cells suchthat the antisense RNA or DNA may be expressed in vivo to inhibitproduction of the PRO10282 polypeptide. When antisense DNA is used,oligodeoxyribonucleotides derived from the translation-initiation site,e.g., between about −10 and +10 positions of the target gene nucleotidesequence, are preferred.

Potential antagonists include small molecules that bind to the activesite, the receptor binding site, or growth factor or other relevantbinding site of the PRO10282 polypeptide, thereby blocking the normalbiological activity of the PRO10282 polypeptide. Examples of smallmolecules include, but are not limited to, small peptides orpeptide-like molecules, preferably soluble peptides, and syntheticnon-peptidyl organic or inorganic compounds.

Ribozymes are enzymatic RNA molecules capable of catalyzing the specificcleavage of RNA. Ribozymes act by sequence-specific hybridization to thecomplementary target RNA, followed by endonucleolytic cleavage. Specificribozyme cleavage sites within a potential RNA target can be identifiedby known techniques. For further details see, e.g., Rossi, CurrentBiology, 4:469-471 (1994), and PCT publication No. WO 97/33551(published Sep. 18, 1997).

Nucleic acid molecules in triple-helix formation used to inhibittranscription should be single-stranded and composed ofdeoxynucleotides. The base composition of these oligonucleotides isdesigned such that it promotes triple-helix formation via Hoogsteenbase-pairing rules, which generally require sizeable stretches ofpurines or pyrimidines on one strand of a duplex. For further detailssee, e.g., PCT publication No. WO 97/33551, supra.

These small molecules can be identified by any one or more of thescreening assays discussed hereinabove and/or by any other screeningtechniques well known for those skilled in the art.

Stra6, a murine retinoic acid inducible gene, has a spermatogeniccycle-dependent expression in Sertoli cells in testis. Based on sequencehomology with Stra6, the PRO10282 polypeptides disclosed herein may havean important role in spermatogenesis, and therefore has a potential usein fertility treatment. It is currently believed that the expression ofPRO10282 polypeptide, just like that of its murine homolog Stra6, isinduced in response to retinoic acid. Therefore, the expression ofPRO10282 at the level of mRNA, by hybridization, or protein, byimmunological assays, can be used to determine whether a test compoundhas a potential for the treatment of a wide variety of retinoidresponsive diseases. Among the types of diseases contemplated astherapeutic targets include psoriasis, acne, dysplasias, cancers andautoimmune diseases. The category of contemplated dysplasias includesprecancerous lesions of the epithelial tissues such as oralleukoplakias, dysplasia of the cervix, larynx and bronchi. The categoryof contemplated cancers includes carcinomas of colon (adenocarcinoma),lung (carcinoma and adenocarcinoma), skin, head and neck, cervix,uterus, breast and prostate. The category of contemplated autoimmunediseases includes rheumatoid arthritis, osteoarthritis, systemic lupuserythematosus, pemphigus valgaris and pemphigus foliaceous. Potentialtherapeutics to treat such diseases and conditions are antibodies andantagonists (including small molecules) of PRO10282.

M. Compositions and Methods for the Treatment of Tumors

The compositions useful in the treatment of tumors associated with theamplification of the genes identified herein include, withoutlimitation, antibodies, small organic and inorganic molecules, peptides,phosphopeptides, antisense and ribozyme molecules, triple helixmolecules, etc., that inhibit the expression and/or activity of thetarget gene product.

For example, antisense RNA and RNA molecules act to directly block thetranslation of mRNA by hybridizing to targeted mRNA and preventingprotein translation. When antisense DNA is used,oligodeoxyribonucleotides derived from the translation initiation site,e.g., between about −10 and +10 positions of the target gene nucleotidesequence, are preferred.

Ribozymes are enzymatic RNA molecules capable of catalyzing the specificcleavage of RNA. Ribozymes act by sequence-specific hybridization to thecomplementary target RNA, followed by endonucleolytic cleavage. Specificribozyme cleavage sites within a potential RNA target can be identifiedby known techniques. For further details see, e.g., Rossi, CurrentBiology, 4:469-471 (1994), and PCT publication No. WO 97/33551(published Sep. 18, 1997).

Nucleic acid molecules in triple helix formation used to inhibittranscription should be single-stranded and composed ofdeoxynucleotides. The base composition of these oligonucleotides isdesigned such that it promotes triple helix formation via Hoogsteen basepairing rules, which generally require sizeable stretches of purines orpyrimidines on one strand of a duplex. For further details see, e.g.,PCT publication No. WO 97/33551, supra.

These molecules can be identified by any or any combination of thescreening assays discussed hereinabove and/or by any other screeningtechniques well known for those skilled in the art.

N. Anti-PRO10282 (Anti-Stra6) Antibodies

The present invention further provides anti-PRO10282 (anti-Stra6)antibodies. Exemplary antibodies include polyclonal, monoclonal,humanized, bispecific, and heteroconjugate antibodies.

1. Polyclonal Antibodies

The anti-PRO10282 antibodies may comprise polyclonal antibodies. Methodsof preparing polyclonal antibodies are known to the skilled artisan.Polyclonal antibodies can be raised in a mammal, for example, by one ormore injections of an immunizing agent and, if desired, an adjuvant.Typically, the immunizing agent and/or adjuvant will be injected in themammal by multiple subcutaneous or intraperitoneal injections. Theimmunizing agent may include the PRO10282 polypeptide or a fusionprotein thereof. It may be useful to conjugate the immunizing agent to aprotein known to be immunogenic in the mammal being immunized. Examplesof such immunogenic proteins include but are not limited to keyholelimpet hemocyanin, serum albumin, bovine thyroglobulin, and soybeantrypsin inhibitor. Examples of adjuvants which may be employed includeFreund's complete adjuvant and MPL-TDM adjuvant (monophosphoryl Lipid A,synthetic trehalose dicorynomycolate). The immunization protocol may beselected by one skilled in the art without undue experimentation.

2. Monoclonal Antibodies

The anti-PRO10282 antibodies may, alternatively, be monoclonalantibodies. Monoclonal antibodies may be prepared using hybridomamethods, such as those described by Kohler and Milstein, Nature, 256:495(1975). In a hybridoma method, a mouse, hamster, or other appropriatehost animal, is typically immunized with an immunizing agent to elicitlymphocytes that produce or are capable of producing antibodies thatwill specifically bind to the immunizing agent. Alternatively, thelymphocytes may be immunized in vitro.

The immunizing agent will typically include the PRO10282 polypeptide ora fusion protein thereof. Generally, either peripheral blood lymphocytes(“PBLs”) are used if cells of human origin are desired, or spleen cellsor lymph node cells are used if non-human mammalian sources are desired.The lymphocytes are then fused with an immortalized cell line using asuitable fusing agent, such as polyethylene glycol, to form a hybridomacell [Goding, Monoclonal Antibodies: Principles and Practice, AcademicPress, (1986) pp. 59-103]. Immortalized cell lines are usuallytransformed mammalian cells, particularly myeloma cells of rodent,bovine and human origin. Usually, rat or mouse myeloma cell lines areemployed. The hybridoma cells may be cultured in a suitable culturemedium that preferably contains one or more substances that inhibit thegrowth or survival of the unfused, immortalized cells. For example, ifthe parental cells lack the enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), the culture medium for the hybridomastypically will include hypoxanthine, aminopterin, and thymidine (“HATmedium”), which substances prevent the growth of HGPRT-deficient cells.

Preferred immortalized cell lines are those that fuse efficiently,support stable high level expression of antibody by the selectedantibody-producing cells, and are sensitive to a medium such as HATmedium. More preferred immortalized cell lines are murine myeloma lines,which can be obtained, for instance, from the Salk Institute CellDistribution Center, San Diego, Calif. and the American Type CultureCollection, Manassas, Va. Human myeloma and mouse-human heteromyelomacell lines also have been described for the production of humanmonoclonal antibodies [Kozbor, J. Immunol., 133:3001 (1984); Brodeur etal., Monoclonal Antibody Production Techniques and Applications, MarcelDekker, Inc., New York, (1987) pp. 51-63].

The culture medium in which the hybridoma cells are cultured can then beassayed for the presence of monoclonal antibodies directed againstPRO10282. Preferably, the binding specificity of monoclonal antibodiesproduced by the hybridoma cells is determined by immunoprecipitation orby an in vitro binding assay, such as radioimmunoassay (RIA) orenzyme-linked immunoabsorbent assay (ELISA). Such techniques and assaysare known in the art. The binding affinity of the monoclonal antibodycan, for example, be determined by the Scatchard analysis of Munson andPollard, Anal. Biochem., 107:220 (1980).

After the desired hybridoma cells are identified, the clones may besubcloned by limiting dilution procedures and grown by standard methods[Goding, supra]. Suitable culture media for this purpose include, forexample, Dulbecco's Modified Eagle's Medium and RPMI-1640 medium.Alternatively, the hybridoma cells may be grown in vivo as ascites in amammal.

The monoclonal antibodies secreted by the subclones may be isolated orpurified from the culture medium or ascites fluid by conventionalimmunoglobulin purification procedures such as, for example, proteinA-Sepharose, hydroxylapatite chromatography, gel electrophoresis,dialysis, or affinity chromatography.

The monoclonal antibodies may also be made by recombinant DNA methods,such as those described in U.S. Pat. No. 4,816,567. DNA encoding themonoclonal antibodies of the invention can be readily isolated andsequenced using conventional procedures (e.g., by using oligonucleotideprobes that are capable of binding specifically to genes encoding theheavy and light chains of murine antibodies). The hybridoma cells of theinvention serve as a preferred source of such DNA. Once isolated, theDNA may be placed into expression vectors, which are then transfectedinto host cells such as simian COS cells, Chinese hamster ovary (CHO)cells, or myeloma cells that do not otherwise produce immunoglobulinprotein, to obtain the synthesis of monoclonal antibodies in therecombinant host cells. The DNA also may be modified, for example, bysubstituting the coding sequence for human heavy and light chainconstant domains in place of the homologous murine sequences [U.S. Pat.No. 4,816,567; Morrison et al., supra] or by covalently joining to theimmunoglobulin coding sequence all or part of the coding sequence for anon-immunoglobulin polypeptide. Such a non-immunoglobulin polypeptidecan be substituted for the constant domains of an antibody of theinvention, or can be substituted for the variable domains of oneantigen-combining site of an antibody of the invention to create achimeric bivalent antibody.

The antibodies may be monovalent antibodies. Methods for preparingmonovalent antibodies are well known in the art. For example, one methodinvolves recombinant expression of immunoglobulin light chain andmodified heavy chain. The heavy chain is truncated generally at anypoint in the Fc region so as to prevent heavy chain crosslinking.Alternatively, the relevant cysteine residues are substituted withanother amino acid residue or are deleted so as to prevent crosslinking.

In vitro methods are also suitable for preparing monovalent antibodies.Digestion of antibodies to produce fragments thereof, particularly, Fabfragments, can be accomplished using routine techniques known in theart.

3. Human and Humanized Antibodies

The anti-PRO10282 antibodies of the invention may further comprisehumanized antibodies or human antibodies. Humanized forms of non-human(e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulinchains or fragments thereof (such as Fv, Fab, Fab′, F(ab′)₂ or otherantigen-binding subsequences of antibodies) which contain minimalsequence derived from non-human immunoglobulin. Humanized antibodiesinclude human immunoglobulins (recipient antibody) in which residuesfrom a complementary determining region (CDR) of the recipient arereplaced by residues from a CDR of a non-human species (donor antibody)such as mouse, rat or rabbit having the desired specificity, affinityand capacity. In some instances, Fv framework residues of the humanimmunoglobulin are replaced by corresponding non-human residues.Humanized antibodies may also comprise residues which are found neitherin the recipient antibody nor in the imported CDR or frameworksequences. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the CDR regions correspond to thoseof a non-human immunoglobulin and all or substantially all of the FRregions are those of a human immunoglobulin consensus sequence. Thehumanized antibody optimally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin [Jones et al., Nature, 321:522-525 (1986); Riechmann etal., Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol.,2:593-596 (1992)].

Methods for humanizing non-human antibodies are well known in the art.Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source which is non-human. These non-humanamino acid residues are often referred to as “import” residues, whichare typically taken from an “import” variable domain. Humanization canbe essentially performed following the method of Winter and co-workers[Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature,332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)], bysubstituting rodent CDRs or CDR sequences for the correspondingsequences of a human antibody. Accordingly, such “humanized” antibodiesare chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantiallyless than an intact human variable domain has been substituted by thecorresponding sequence from a non-human species. In practice, humanizedantibodies are typically human antibodies in which some CDR residues andpossibly some FR residues are substituted by residues from analogoussites in rodent antibodies.

Human antibodies can also be produced using various techniques known inthe art, including phage display libraries [Hoogenboom and Winter, J.Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581(1991)]. The techniques of Cole et al. and Boerner et al. are alsoavailable for the preparation of human monoclonal antibodies (Cole etal., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77(1985) and Boerner et al., J. Immunol., 147(1):86-95 (1991)]. Similarly,human antibodies can be made by introducing of human immunoglobulin lociinto transgenic animals, e.g., mice in which the endogenousimmunoglobulin genes have been partially or completely inactivated. Uponchallenge, human antibody production is observed, which closelyresembles that seen in humans in all respects, including generearrangement, assembly, and antibody repertoire. This approach isdescribed, for example, in U.S. Pat. Nos. 5,545,807; 5,545,806;5,569,825; 5,625,126; 5,633,425; 5,661,016, and in the followingscientific publications: Marks et al., Bio/Technology 10, 779-783(1992); Lonberg et al., Nature 368 856-859 (1994); Morrison, Nature 368,812-13 (1994); Fishwild et al., Nature Biotechnology 14, 845-51 (1996);Neuberger, Nature Biotechnology 14, 826 (1996); Lonberg and Huszar,Intern. Rev. Immunol. 13 65-93 (1995).

4. Bispecific Antibodies

Bispecific antibodies are monoclonal, preferably human or humanized,antibodies that have binding specificities for at least two differentantigens. In the present case, one of the binding specificities is forthe PRO10282, the other one is for any other antigen, and preferably fora cell-surface protein or receptor or receptor subunit.

Methods for making bispecific antibodies are known in the art.Traditionally, the recombinant production of bispecific antibodies isbased on the co-expression of two immunoglobulin heavy-chain/light-chainpairs, where the two heavy chains have different specificities [Milsteinand Cuello, Nature, 305:537-539 (1983)]. Because of the randomassortment of immunoglobulin heavy and light chains, these hybridomas(quadromas) produce a potential mixture of ten different antibodymolecules, of which only one has the correct bispecific structure. Thepurification of the correct molecule is usually accomplished by affinitychromatography steps. Similar procedures are disclosed in WO 93/08829,published 13 May 1993, and in Traunecker et al., EMBO J., 10:3655-3659(1991).

Antibody variable domains with the desired binding specificities(antibody-antigen combining sites) can be fused to immunoglobulinconstant domain sequences. The fusion preferably is with animmunoglobulin heavy-chain constant domain, comprising at least part ofthe hinge, CH2, and CH3 regions. It is preferred to have the firstheavy-chain constant region (CH1) containing the site necessary forlight-chain binding present in at least one of the fusions. DNAsencoding the immunoglobulin heavy-chain fusions and, if desired, theimmunoglobulin light chain, are inserted into separate expressionvectors, and are co-transfected into a suitable host organism. Forfurther details of generating bispecific antibodies see, for example,Suresh et al., Methods in Enzymology, 121:210 (1986).

According to another approach described in WO 96/27011, the interfacebetween a pair of antibody molecules can be engineered to maximize thepercentage of heterodimers which are recovered from recombinant cellculture. The preferred interface comprises at least a part of the CH3region of an antibody constant domain. In this method, one or more smallamino acid side chains from the interface of the first antibody moleculeare replaced with larger side chains (e.g. tyrosine or tryptophan).Compensatory “cavities” of identical or similar size to the large sidechain(s) are created on the interface of the second antibody molecule byreplacing large amino acid side chains with smaller ones (e.g. alanineor threonine). This provides a mechanism for increasing the yield of theheterodimer over other unwanted end-products such as homodimers.

Bispecific antibodies can be prepared as full length antibodies orantibody fragments (e.g. F(ab′)₂ bispecific antibodies). Techniques forgenerating bispecific antibodies from antibody fragments have beendescribed in the literature. For example, bispecific antibodies can beprepared can be prepared using chemical linkage. Brennan et al., Science229:81 (1985) describe a procedure wherein intact antibodies areproteolytically cleaved to generate F(ab′)₂ fragments. These fragmentsare reduced in the presence of the dithiol complexing agent sodiumarsenite to stabilize vicinal dithiols and prevent intermoleculardisulfide formation. The Fab′ fragments generated are then converted tothionitrobenzoate (TNB) derivatives. One of the Fab′-TNB derivatives isthen reconverted to the Fab′-thiol by reduction with mercaptoethylamineand is mixed with an equimolar amount of the other Fab′-TNB derivativeto form the bispecific antibody. The bispecific antibodies produced canbe used as agents for the selective immobilization of enzymes.

Fab′ fragments may be directly recovered from E. coli and chemicallycoupled to form bispecific antibodies. Shalaby et al., J. Exp. Med.175:217-225 (1992) describe the production of a fully humanizedbispecific antibody F(ab′)₂ molecule. Each Fab′ fragment was separatelysecreted from E. coli and subjected to directed chemical coupling invitro to form the bispecific antibody. The bispecific antibody thusformed was able to bind to cells overexpressing the ErbB2 receptor andnormal human T cells, as well as trigger the lytic activity of humancytotoxic lymphocytes against human breast tumor targets.

Various technique for making and isolating bispecific antibody fragmentsdirectly from recombinant cell culture have also been described. Forexample, bispecific antibodies have been produced using leucine zippers.Kostelny et al., J. Immunol. 148(5): 1547-1553 (1992). The leucinezipper peptides from the Fos and Jun proteins were linked to the Fab′portions of two different antibodies by gene fusion. The antibodyhomodimers were reduced at the hinge region to form monomers and thenre-oxidized to form the antibody heterodimers. This method can also beutilized for the production of antibody homodimiers. The “diabody”technology described by Hollinger et al., Proc. Natl. Acad. Sci. USA90:6444-6448 (1993) has provided an alternative mechanism for makingbispecific antibody fragments. The fragments comprise a heavy-chainvariable domain (V_(H)) connected to a light-chain variable domain(V_(L)) by a linker which is too short to allow pairing between the twodomains on the same chain. Accordingly, the V_(H) and V_(L) domains ofone fragment are forced to pair with the complementary V_(L) and V_(H)domains of another fragment, thereby forming two antigen-binding sites.Another strategy for making bispecific antibody fragments by the use ofsingle-chain Fv (sFv) dimers has also been reported. See, Gruber et al.,J. Immunol. 152:5368 (1994). Antibodies with more than two valencies arecontemplated. For example, trispecific antibodies can be prepared. Tuttet al., J. Immunol. 147:60 (1991).

Exemplary bispecific antibodies may bind to two different epitopes on agiven PRO10282 polypeptide herein. Alternatively, an anti-PRO10282polypeptide arm may be combined with an arm which binds to a triggeringmolecule on a leukocyte such as a T-cell receptor molecule (e.g. CD2,CD3, CD28, or B7), or Fc receptors for IgG (Fc R), such as Fc RI (CD64),Fc RII (CD32) and Fc RIII (CD16) so as to focus cellular defensemechanisms to the cell expressing the particular PRO10282 polypeptide.Bispecific antibodies may also be used to localize cytotoxic agents tocells which express a particular PRO10282 polypeptide. These antibodiespossess a PRO10282-binding arm and an arm which binds a cytotoxic agentor a radionuclide chelator, such as EOTUBE, DPTA, DOTA, or TETA. Anotherbispecific antibody of interest binds the PRO10282 polypeptide andfurther binds tissue factor (TF).

5. Heteroconjugate Antibodies

Heteroconjugate antibodies are also within the scope of the presentinvention. Heteroconjugate antibodies are composed of two covalentlyjoined antibodies. Such antibodies have, for example, been proposed totarget immune system cells to unwanted cells [U.S. Pat. No. 4,676,980],and for treatment of HIV infection [WO 91/00360; WO 92/200373; EP03089]. It is contemplated that the antibodies may be prepared in vitrousing known methods in synthetic protein chemistry, including thoseinvolving crosslinking agents. For example, immunotoxins may beconstructed using a disulfide exchange reaction or by forming athioether bond. Examples of suitable reagents for this purpose includeiminothiolate and methyl-4-mercaptobutyrimidate and those disclosed, forexample, in U.S. Pat. No. 4,676,980.

6. Effector Function Engineering

It may be desirable to modify the antibody of the invention with respectto effector function, so as to enhance, e.g., the effectiveness of theantibody in treating cancer. For example, cysteine residue(s) may beintroduced into the Fc region, thereby allowing interchain disulfidebond formation in this region. The homodimeric antibody thus generatedmay have improved internalization capability and/or increasedcomplement-mediated cell killing and antibody-dependent cellularcytotoxicity (ADCC). See Caron et al., J. Exp Med., 176: 1191-1195(1992) and Shopes, J. Immunol., 148: 2918-2922 (1992). Homodimericantibodies with enhanced anti-tumor activity may also be prepared usingheterobifunctional cross-linkers as described in Wolff et al. CancerResearch, 53: 2560-2565 (1993). Alternatively, an antibody can beengineered that has dual Fc regions and may thereby have enhancedcomplement lysis and ADCC capabilities. See Stevenson et al.,Anti-Cancer Drug Design. 3: 219-230 (1989).

7. Immunoconjugates

The invention also pertains to immunoconjugates comprising an antibodyconjugated to a cytotoxic agent such as a chemotherapeutic agent, toxin(e.g., an enzymatically active toxin of bacterial, fungal, plant, oranimal origin, or fragments thereof), or a radioactive isotope (i.e., aradioconjugate).

Chemotherapeutic agents useful in the generation of suchimmunoconjugates have been described above. Enzymatically active toxinsand fragments thereof that can be used include diphtheria A chain,nonbinding active fragments of diphtheria toxin, exotoxin A chain (fromPseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain,alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolacaamericana proteins (PAPI, PAPII, and PAP-S), momordica charantiainhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin,mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes. Avariety of radionuclides are available for the production ofradioconjugated antibodies. Examples include ²¹²Bi, ¹³¹I, ¹³¹In, ⁹⁰Y,and ¹⁸⁶Re.

Conjugates of the antibody and cytotoxic agent are made using a varietyof bifunctional protein-coupling agents such asN-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane(IT), bifunctional derivatives of imidoesters (such as dimethyladipimidate HCL), active esters (such as disuccinimidyl suberate),aldehydes (such as glutareldehyde), bis-azido compounds (such as bis(p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such asbis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin canbe prepared as described in Vitetta et al., Science, 238: 1098 (1987).Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the antibody. See WO94/11026.

In another embodiment, the antibody may be conjugated to a “receptor”(such streptavidin) for utilization in tumor pretargeting wherein theantibody-receptor conjugate is administered to the patient, followed byremoval of unbound conjugate from the circulation using a clearing agentand then administration of a “ligand” (e.g., avidin) that is conjugatedto a cytotoxic agent (e.g., a radionucleotide).

8. Immunoliposomes

The antibodies disclosed herein may also be formulated asimmunoliposomes. Liposomes containing the antibody are prepared bymethods known in the art, such as described in Epstein et al., Proc.Natl. Acad. Sci. USA, 82: 3688 (1985); Hwang et al., Proc. Natl. Acad.Sci. USA, 77: 4030 (1980); and U.S. Pat. Nos. 4,485,045 and 4,544,545.Liposomes with enhanced circulation time are disclosed in U.S. Pat. No.5,013,556.

Particularly useful liposomes can be generated by the reverse-phaseevaporation method with a lipid composition comprisingphosphatidylcholine, cholesterol, and PEG-derivatizedphosphatidylethanolamine (PEG-PE). Liposomes are extruded throughfilters of defined pore size to yield liposomes with the desireddiameter. Fab′ fragments of the antibody of the present invention can beconjugated to the liposomes as described in Martin et al., J. Biol.Chem., 257: 286-288 (1982) via a disulfide-interchange reaction. Achemotherapeutic agent (such as Doxorubicin) is optionally containedwithin the liposome. See Gabizon et al., J. National Cancer Inst.,81(19): 1484 (1989).

8. Antibody-Dependent Enzyme Mediated Prodrug Therapy (ADEPT)

The antibodies of the present invention may also be used in ADEPT byconjugating the antibody to a prodrug-activating enzyme which converts aprodrug (e.g., a peptidyl chemotherapeutic agent, see WO 81/01145) to anactive anti-cancer drug. See, for example, WO 88/07378 and U.S. Pat. No.4,975,278.

The enzyme component of the immunoconjugate useful for ADEPT includesany enzyme capable of acting on a prodrug in such as way so as toconvert it into its more active, cytotoxic form.

Enzymes that are useful in the method of this invention include, but arenot limited to, glycosidase, glucose oxidase, human lysosyme, humanglucuronidase, alkaline phosphatase useful for convertingphosphate-containing prodrugs into free drugs; arylsulfatase useful forconverting sulfate-containing prodrugs into free drugs; cytosinedeaminase useful for converting non-toxic 5-fluorocytosine into theanti-cancer drug 5-fluorouracil; proteases, such as serratia protease,thermolysin, subtilisin, carboxypeptidases (e.g., carboxypeptidase G2and carboxypeptidase A) and cathepsins (such as cathepsins B and L),that are useful for converting peptide-containing prodrugs into freedrugs; D-alanylcarboxypeptidases, useful for converting prodrugs thatcontain D-amino acid substituents; carbohydrate-cleaving enzymes such as-galactosidase and neuraminidase useful for converting glycosylatedprodrugs into free drugs; -lactamase useful for converting drugsderivatized with -lactams into free drugs; and penicillin amidases, suchas penicillin Vamidase or penicillin G amidase, useful for convertingdrugs derivatized at their amine nitrogens with phenoxyacetyl orphenylacetyl groups, respectively, into free drugs. Alternatively,antibodies with enzymatic activity, also known in the art as “abzymes”can be used to convert the prodrugs of the invention into free activedrugs (see, e.g., Massey, Nature, 328:457-458 (1987)). Antibody-abzymeconjugates can be prepared as described herein for delivery of theabzyme to a tumor cell population.

The enzymes of this invention can be covalently bound to theanti-PRO10282 (anti-Stra6) antibodies by techniques well known in theart such as the use of the heterobifunctional cross-linking agentsdiscussed above. Alternatively, fusion proteins comprising at least theantigen binding region of the antibody of the invention linked to atleast a functionally active portion of an enzyme of the invention can beconstructed using recombinant DNA techniques well known in the art (see,e.g., Neuberger et al., Nature, 312:604-608 (1984)).

10. Pharmaceutical Compositions of Antibodies

Antibodies specifically binding a PRO10282 polypeptide identifiedherein, as well as other molecules identified by the screening assaysdisclosed hereinbefore, can be administered for the treatment of variousdisorders in the form of pharmaceutical compositions.

If the PRO10282 polypeptide is intracellular and whole antibodies areused as inhibitors, internalizing antibodies are preferred. However,lipofections or liposomes can also be used to deliver the antibody, oran antibody fragment, into cells. Where antibody fragments are used, thesmallest inhibitory fragment that specifically binds to the bindingdomain of the target protein is preferred. For example, based upon thevariable-region sequences of an antibody, peptide molecules can bedesigned that retain the ability to bind the target protein sequence.Such peptides can be synthesized chemically and/or produced byrecombinant DNA technology. See, e.g., Marasco et al., Proc. Natl. Acad.Sci. USA, 90: 7889-7893 (1993). The formulation herein may also containmore than one active compound as necessary for the particular indicationbeing treated, preferably those with complementary activities that donot adversely affect each other. Alternatively, or in addition, thecomposition may comprise an agent that enhances its function, such as,for example, a cytotoxic agent, cytokine, chemotherapeutic agent, orgrowth-inhibitory agent. Such molecules are suitably present incombination in amounts that are effective for the purpose intended.

The active ingredients may also be entrapped in microcapsules prepared,for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsules and poly-(methylmethacylate) microcapsules,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles, andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences, supra.

The formulations to be used for in vivo administration must be sterile.This is readily accomplished by filtration through sterile filtrationmembranes.

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the antibody, which matrices are in theform of shaped articles, e.g., films, or microcapsules. Examples ofsustained-release matrices include polyesters, hydrogels (for example,poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides(U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid andethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradablelactic acid-glycolic acid copolymers such as the LUPRON DEPOT™(injectable microspheres composed of lactic acid-glycolic acid copolymerand leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid. Whilepolymers such as ethylene-vinyl acetate and lactic acid-glycolic acidenable release of molecules for over 100 days, certain hydrogels releaseproteins for shorter time periods. When encapsulated antibodies remainin the body for a long time, they may denature or aggregate as a resultof exposure to moisture at 37° C., resulting in a loss of biologicalactivity and possible changes in immunogenicity. Rational strategies canbe devised for stabilization depending on the mechanism involved. Forexample, if the aggregation mechanism is discovered to be intermolecularS—S bond formation through thio-disulfide interchange, stabilization maybe achieved by modifying sulfhydryl residues, lyophilizing from acidicsolutions, controlling moisture content, using appropriate additives,and developing specific polymer matrix compositions.

O. Uses for Anti-PRO10282 (Anti-Stra6) Antibodies

The anti-PRO10282 antibodies of the invention have various utilities.For example, anti-PRO10282 antibodies may be used in diagnostic assaysfor PRO10282, e.g., detecting its expression in specific cells, tissues,or serum. Various diagnostic assay techniques known in the art may beused, such as competitive binding assays, direct or indirect sandwichassays and immunoprecipitation assays conducted in either heterogeneousor homogeneous phases [Zola, Monoclonal Antibodies: A Manual ofTechniques, CRC Press, Inc. (1987) pp. 147-158]. The antibodies used inthe diagnostic assays can be labeled with a detectable moiety. Thedetectable moiety should be capable of producing, either directly orindirectly, a detectable signal. For example, the detectable moiety maybe a radioisotope, such as ³H, ¹⁴C, ³²P, ³⁵S, or ¹²⁵I, a fluorescent orchemiluminescent compound, such as fluorescein isothiocyanate,rhodamine, or luciferin, or an enzyme, such as alkaline phosphatase,beta-galactosidase or horseradish peroxidase. Any method known in theart for conjugating the antibody to the detectable moiety may beemployed, including those methods described by Hunter et al., Nature,144:945 (1962); David et al., Biochemistry, 13:1014 (1974); Pain et al.,J. Immunol. Meth., 40:219 (1981); and Nygren, J. Histochem. andCytochem., 30:407 (1982).

Anti-PRO10282 antibodies also are useful for the affinity purificationof PRO10282 from recombinant cell culture or natural sources. In thisprocess, the antibodies against PRO10282 are immobilized on a suitablesupport, such a Sephadex resin or filter paper, using methods well knownin the art. The immobilized antibody then is contacted with a samplecontaining the PRO10282 to be purified, and thereafter the support iswashed with a suitable solvent that will remove substantially all thematerial in the sample except the PRO10282, which is bound to theimmobilized antibody. Finally, the support is washed with anothersuitable solvent that will release the PRO10282 from the antibody.

Anti-PRO10282 antibodies can be used to monitor the expression ofPRO10282 polypeptide which has a potential use as a sensitive method toscreen for therapeutically useful compounds against a wide variety ofretinoid responsive diseases. Among the types of diseases contemplatedas therapeutic targets include psoriasis, acne, dysplasias, cancers andautoimmune diseases. The category of contemplated dysplasias includesprecancerous lesions of the epithelial tissues such as oralleukoplakias, dysplasia of the cervix, larynx and bronchi. The categoryof contemplated cancers includes carcinomas of colon (adenocarcinoma),lung (adenocarcinoma and carcinoma), skin, head and neck, cervix,uterus, breast and prostate. The category of contemplated autoimmunediseases includes rheumatoid arthritis, osteoarthritis, systemic lupuserythematosus, pemphigus valgaris and pemphigus foliaceous.Anti-PRO10282 antibodies have potential therapeutic use in the treatmentof such diseases and conditions.

P. Methods of Treatment Using Anti-PRO10282 (Stra6) Antibodies and OtherStra6 Antagonists

It is contemplated that the antibodies and other anti-tumor compounds ofthe present invention may be used to treat various conditions, includingthose characterized by overexpression and/or activation of the amplifiedgenes identified herein. Exemplary conditions or disorders to be treatedwith such antibodies and other compounds, including, but not limited to,small organic and inorganic molecules, peptides, antisense molecules,etc., include benign or malignant tumors (e.g., renal, liver, kidney,bladder, breast, gastric, ovarian, colorectal, prostate, pancreatic,lung, vulval, thyroid, hepatic carcinomas; sarcomas; glioblastomas; andvarious head and neck tumors); leukemias and lymphoid malignancies;other disorders such as neuronal, glial, astrocytal, hypothalamic andother glandular, macrophagal, epithelial, stromal and blastocoelicdisorders; and inflammatory, angiogenic and immunologic disorders.Particularly preferred targets for treatment with anti-Stra6 antibodiesand other antagonists of the present invention are tumors that harborgenetic defects in the Wnt-1 pathway (e.g. leading to abnormalactivation of β-catenin signaling) and/or overexpress Stra6. It has beenfound that human cancers harboring genetic defects in the Wnt-1 pathwaytypically also exhibit overexpression of Stra6, but not all Stra6overexpressing tumors have been known to be associated with mutations inthe Wnt-1 pathway. The Stra6 antagonists of the present invention havegreat potential in the treatment of Stra6 overexpressing tumors. Apreferred group of such tumors includes colorectal tumors, ovary,endometrium, and Wilm's kidney tumors, melanoma, and pheochromocytome (atumor derived from the adrenal medulla).

The anti-tumor agents of the present invention, e.g., antibodies, areadministered to a mammal, preferably a human, in accord with knownmethods, such as intravenous administration as a bolus or by continuousinfusion over a period of time, by intramuscular, intraperitoneal,intracerobrospinal, subcutaneous, intra-articular, intrasynovial,intrathecal, oral, topical, or inhalation routes. Intravenousadministration of the antibody is preferred.

Other therapeutic regimens may be combined with the administration ofthe anti-cancer agents, e.g., antibodies of the instant invention. Forexample, the patient to be treated with such anti-cancer agents may alsoreceive radiation therapy. Alternatively, or in addition, achemotherapeutic agent may be administered to the patient. Preparationand dosing schedules for such chemotherapeutic agents may be usedaccording to manufacturers' instructions or as determined empirically bythe skilled practitioner. Preparation and dosing schedules for suchchemotherapy are also described in Chemotherapy Service Ed., M. C.Perry, Williams & Wilkins, Baltimore, Md. (1992). The chemotherapeuticagent may precede, or follow administration of the anti-tumor agent,e.g., antibody, or may be given simultaneously therewith. The antibodymay be combined with an anti-estrogen compound such as tamoxifen or ananti-progesterone such as onapristone (see, EP 616812) in dosages knownfor such molecules.

It may be desirable to also administer antibodies against other tumorassociated antigens, such as antibodies which bind to the ErbB2, EGFR,ErbB3, ErbB4, or vascular endothelial factor (VEGF). Alternatively, orin addition, two or more antibodies binding the same or two or moredifferent antigens disclosed herein may be co-administered to thepatient. Sometimes, it may be beneficial to also administer one or morecytokines to the patient. In a preferred embodiment, the antibodiesherein are co-administered with a growth inhibitory agent. For example,the growth inhibitory agent may be administered first, followed by anantibody of the present invention. However, simultaneous administrationor administration of the antibody of the present invention first is alsocontemplated. Suitable dosages for the growth inhibitory agent are thosepresently used and may be lowered due to the combined action (synergy)of the growth inhibitory agent and the antibody herein.

For the prevention or treatment of disease, the appropriate dosage of ananti-tumor agent, e.g., an antibody herein will depend on the type ofdisease to be treated, as defined above, the severity and course of thedisease, whether the agent is administered for preventive or therapeuticpurposes, previous therapy, the patient's clinical history and responseto the agent, and the discretion of the attending physician. The agentis suitably administered to the patient at one time or over a series oftreatments.

For example, depending on the type and severity of the disease, about 1g/kg to 15 mg/kg (e.g., 0.1-20 mg/kg) of antibody is an initialcandidate dosage for administration to the patient, whether, forexample, by one or more separate administrations, or by continuousinfusion. A typical daily dosage might range from about 1 g/kg to 100mg/kg or more, depending on the factors mentioned above. For repeatedadministrations over several days or longer, depending on the condition,the treatment is sustained until a desired suppression of diseasesymptoms occurs. However, other dosage regimens may be useful. Theprogress of this therapy is easily monitored by conventional techniquesand assays.

Q. Articles of Manufacture

In another embodiment of the invention, an article of manufacturecontaining materials useful for the diagnosis or treatment of thedisorders described above is provided. The article of manufacturecomprises a container and a label. Suitable containers include, forexample, bottles, vials, syringes, and test tubes. The containers may beformed from a variety of materials such as glass or plastic. Thecontainer holds a composition which is effective for diagnosing ortreating the condition and may have a sterile access port (for examplethe container may be an intravenous solution bag or a vial having astopper pierceable by a hypodermic injection needle). The active agentin the composition is usually an anti-tumor agent capable of interferingwith the activity of a gene product identified herein, e.g., anantibody. The label on, or associated with, the container indicates thatthe composition is used for diagnosing or treating the condition ofchoice. The article of manufacture may further comprise a secondcontainer comprising a pharmaceutically-acceptable buffer, such asphosphate-buffered saline, Ringer's solution and dextrose solution. Itmay further include other materials desirable from a commercial and userstandpoint, including other buffers, diluents, filters, needles,syringes, and package inserts with instructions for use.

R. Diagnosis and Prognosis of Tumors

While cell surface proteins, such as growth receptors overexpressed incertain tumors are excellent targets for drug candidates or tumor (e.g.,cancer) treatment, the same proteins along with secreted proteinsencoded by the genes amplified in tumor cells find additional use in thediagnosis and prognosis of tumors. For example, antibodies directedagainst the protein products of genes amplified in tumor cells can beused as tumor diagnostics or prognostics.

For example, antibodies, including antibody fragments, can be used toqualitatively or quantitatively detect the expression of proteinsencoded by the amplified genes (“marker gene products”). The antibodypreferably is equipped with a detectable, e.g., fluorescent label, andbinding can be monitored by light microscopy, flow cytometry,fluorimetry, or other techniques known in the art. These techniques areparticularly suitable, if the amplified gene encodes a cell surfaceprotein, e.g., a growth factor. Such binding assays are performedessentially as described in section 5 above.

In situ detection of antibody binding to the marker gene products can beperformed, for example, by immunofluorescence or immunoelectronmicroscopy. For this purpose, a histological specimen is removed fromthe patient, and a labeled antibody is applied to it, preferably byoverlaying the antibody on a biological sample. This procedure alsoallows for determining the distribution of the marker gene product inthe tissue examined It will be apparent for those skilled in the artthat a wide variety of histological methods are readily available for insitu detection.

The following examples are offered for illustrative purposes only, andare not intended to limit the scope of the present invention in any way.

All patent and literature references cited in the present specificationare hereby incorporated by reference in their entirety.

EXAMPLES

Commercially available reagents referred to in the examples were usedaccording to manufacturer's instructions unless otherwise indicated. Thesource of those cells identified in the following examples, andthroughout the specification, by ATCC accession numbers is the AmericanType Culture Collection, Manassas, Va. In the following Examples, unlessotherwise specified, “Sra6” will refer to native sequence PRO10282polypeptide.

Example 1 Isolation of cDNA Clones Encoding a Human PRO10282 andPRO19578 Polypeptides

cDNA clones (DNA148380-2827 and DNA148389-2827-1) encoding native humanPRO10282 and PRO19578 polypeptides were identified using a yeast screen,in a human fetal brain cDNA library that preferentially represents the5′ ends of the primary cDNA clones.

Clone DNA148380-2827 contains a single open reading frame with anapparent translational initiation site at nucleotide positions 49-51 andending at the stop codon at nucleotide positions 2050-2052 (FIG. 1). Thepredicted polypeptide precursor is 667 amino acids long (FIG. 2). Thefull-length PRO10282 protein shown in FIG. 2 has an estimated molecularweight of about 73502 daltons and a pI of about 9.26. Analysis of thefull-length PRO10282 sequence shown in FIG. 2 (SEQ ID NO:2) evidencesthe presence of a variety of important polypeptide domains as shown inFIG. 2, wherein the locations given for those important polypeptidedomains are approximate as described above. Clone DNA148380-2827 hasbeen deposited with ATCC on Jan. 11, 2000 and is assigned ATCC DepositNo. PTA-1181.

Clone DNA148389-2827-1 contains a single open reading frame with anapparent translational initiation site at nucleotide positions 186-188and ending at the stop codon at nucleotide positions 2160-2162 (FIG. 6,SEQ ID NO: 4). The predicted polypeptide precursor is 658 amino acidslong (FIG. 7, SEQ ID NO: 5). The full-length PRO19578 protein shown inFIG. 7 has an estimated molecular weight of about 72583 daltons and a pIof about 9.36. Analysis of the full-length PRO19578 sequence shown inFIG. 7 (SEQ ID NO: 5) evidences the presence of a variety of importantpolypeptide domains as shown in FIG. 7, wherein the locations given forthose important polypeptide domains are approximate as described above.Noteworthy is the presence of nine potential transmembrane domains andfourteen cysteine residues conserved between the human and thecorresponding mouse sequence. While mouse Stra6 has three potentialN-linked glycosylation sites, the human PRO19578 (native human Stra6)polypeptide has one. Clone 148389-2827-1 has been deposited with ATCC onFeb. 23, 2000, and is assigned ATCC Deposit No. PTA-1402.

An analysis of the Dayhoff database (version 35.45 SwissProt 35), usingthe ALIGN-2 sequence alignment analysis of the full-length sequenceshown in FIG. 2 (SEQ ID NO:2), and of the full-length sequence shown inFIG. 7 (SEQ ID NO: 5) evidenced sequence identity between the PRO10282amino acid sequence and the following Dayhoff sequences: AF062476,P_W88559 and HGS_RE259.

As shown in FIG. 8, comparison of the full-length human PRO10282 andPRO19578 polypeptides shows that PRO19578 contains a deletion of nineamino acids (SPVDFLAGD; SEQ ID NO: 13) at positions 89-97 of thePRO10282 amino acid sequence. In addition, PRO19578 contains anisoleucine (1) at amino acid position 518 in place of methionine (M) atthe corresponding position (position 527) of PRO10282, which resultsfrom a G/A polymorphism at this position. Both the PRO10282 and thenative sequence PRO19578 polypeptides are believed to be the humanhomologues of the mouse Stra6 polypeptide, and are, therefore, alsoreferred to as “Stra6.” Mouse Stra6 and the native sequence humanfull-length PRO10282 polypeptide encoded by DNA148340-2827 show about74% amino acid sequence identity.

FIG. 9 shows the hydrophobicity plot of the native sequence humanfull-length PRO10282 polypeptide encoded by DNA148380-2827, brieflyreferred to as “human Stra6.” As shown in FIG. 9, about 50% of the aminoacid residues in this 667 amino acids long polypeptide are hydrophobic.

The human Stra6 gene was localized to chromosome 15q23 as determined byUNIGENE. Preliminary fine mapping indicates that Stra6 is located in theSTS interval D15S124-D15S160 and the GeneMap'98 position corresponds to244.52 on the G3 map.

Example 2 Use of PRO10282 and PRO19578 as a Hybridization Probe

The following method describes use of a nucleotide sequence encodingPRO10282 and PRO19578 as a hybridization probe.

DNA comprising the coding sequence of full-length or mature PRO10282 orPRO19578 is employed as a probe to screen for homologous DNAs (such asthose encoding naturally-occurring variants of PRO10282 or PRO19578) inhuman tissue cDNA libraries or human tissue genomic libraries.

Hybridization and washing of filters containing either library DNAs isperformed under the following high stringency conditions. Hybridizationof radiolabeled PRO10282-derived probe to the filters is performed in asolution of 50% formamide, 5×SSC, 0.1% SDS, 0.1% sodium pyrophosphate,50 mM sodium phosphate, pH 6.8, 2×Denhardt's solution, and 10% dextransulfate at 42° C. for 20 hours. Washing of the filters is performed inan aqueous solution of 0.1×SSC and 0.1% SDS at 42° C.

DNAs having a desired sequence identity with the DNA encodingfull-length native sequence PRO10282 or PRO19578 can then be identifiedusing standard techniques known in the art.

Example 3 Expression of PRO10282 and PRO19578 in E. coli

This example illustrates preparation of an unglycosylated form ofPRO10282 or PRO19578 by recombinant expression in E. coli.

The DNA sequence encoding PRO10282 or PRO19578 is initially amplifiedusing selected PCR primers. The primers should contain restrictionenzyme sites which correspond to the restriction enzyme sites on theselected expression vector. A variety of expression vectors may beemployed. An example of a suitable vector is pBR322 (derived from E.coli; see Bolivar et al., Gene, 2:95 (1977)) which contains genes forampicillin and tetracycline resistance. The vector is digested withrestriction enzyme and dephosphorylated. The PCR amplified sequences arethen ligated into the vector. The vector will preferably includesequences which encode for an antibiotic resistance gene, a trppromoter, a polyhis leader (including the first six STII codons, polyhissequence, and enterokinase cleavage site), the PRO10282 or PRO19578coding region, lambda transcriptional terminator, and an argU gene.

The ligation mixture is then used to transform a selected E. coli strainusing the methods described in Sambrook et al., supra. Transformants areidentified by their ability to grow on LB plates and antibioticresistant colonies are then selected. Plasmid DNA can be isolated andconfirmed by restriction analysis and DNA sequencing.

Selected clones can be grown overnight in liquid culture medium such asLB broth supplemented with antibiotics. The overnight culture maysubsequently be used to inoculate a larger scale culture. The cells arethen grown to a desired optical density, during which the expressionpromoter is turned on.

After culturing the cells for several more hours, the cells can beharvested by centrifugation. The cell pellet obtained by thecentrifugation can be solubilized using various agents known in the art,and the solubilized PRO10282 protein can then be purified using a metalchelating column under conditions that allow tight binding of theprotein.

PRO10282 or PRO19578 may be expressed in E. coli in a poly-His taggedform, using the following procedure. The DNA encoding PRO10282 orPRO19578 is initially amplified using selected PCR primers. The primerswill contain restriction enzyme sites which correspond to therestriction enzyme sites on the selected expression vector, and otheruseful sequences providing for efficient and reliable translationinitiation, rapid purification on a metal chelation column, andproteolytic removal with enterokinase. The PCR-amplified, poly-Histagged sequences are then ligated into an expression vector, which isused to transform an E. coli host based on strain 52 (W3110 fuhA(tonA)lon galE rpoHts(htpRts) clpP(lacIq). Transformants are first grown in LBcontaining 50 mg/ml carbenicillin at 30° C. with shaking until an O.D.600 of 3-5 is reached. Cultures are then diluted 50-100 fold into CRAPmedia (prepared by mixing 3.57 g (NH₄)₂SO₄, 0.71 g sodium citrate 2H2O,1.07 g KCl, 5.36 g Difco yeast extract, 5.36 g Sheffield hycase SF in500 mL water, as well as 110 mM MPOS, pH 7.3, 0.55% (w/v) glucose and 7mM MgSO₄) and grown for approximately 20-30 hours at 30° C. withshaking. Samples are removed to verify expression by SDS-PAGE analysis,and the bulk culture is centrifuged to pellet the cells. Cell pelletsare frozen until purification and refolding.

E. coli paste from 0.5 to 1 L fermentations (6-10 g pellets) isresuspended in 10 volumes (w/v) in 7 M guanidine, 20 mM Tris, pH 8buffer. Solid sodium sulfite and sodium tetrathionate is added to makefinal concentrations of 0.1M and 0.02 M, respectively, and the solutionis stirred overnight at 4° C. This step results in a denatured proteinwith all cysteine residues blocked by sulfitolization. The solution iscentrifuged at 40,000 rpm in a Beckman Ultracentrifuge for 30 min. Thesupernatant is diluted with 3-5 volumes of metal chelate column buffer(6 M guanidine, 20 mM Tris, pH 7.4) and filtered through 0.22 micronfilters to clarify. The clarified extract is loaded onto a 5 ml QiagenNi-NTA metal chelate column equilibrated in the metal chelate columnbuffer. The column is washed with additional buffer containing 50 mMimidazole (Calbiochem, Utrol grade), pH 7.4. The protein is eluted withbuffer containing 250 mM imidazole. Fractions containing the desiredprotein are pooled and stored at 4° C. Protein concentration isestimated by its absorbance at 280 nm using the calculated extinctioncoefficient based on its amino acid sequence.

The proteins are refolded by diluting the sample slowly into freshlyprepared refolding buffer consisting of: 20 mM Tris, pH 8.6, 0.3 M NaCl,2.5 M urea, 5 mM cysteine, 20 mM glycine and 1 mM EDTA. Refoldingvolumes are chosen so that the final protein concentration is between 50to 100 micrograms/ml. The refolding solution is stirred gently at 4° C.for 12-36 hours. The refolding reaction is quenched by the addition ofTFA to a final concentration of 0.4% (pH of approximately 3). Beforefurther purification of the protein, the solution is filtered through a0.22 micron filter and acetonitrile is added to 2-10% finalconcentration. The refolded protein is chromatographed on a Poros R1/Hreversed phase column using a mobile buffer of 0.1% TFA with elutionwith a gradient of acetonitrile from 10 to 80%. Aliquots of fractionswith A280 absorbance are analyzed on SDS polyacrylamide gels andfractions containing homogeneous refolded protein are pooled. Generally,the properly refolded species of most proteins are eluted at the lowestconcentrations of acetonitrile since those species are the most compactwith their hydrophobic interiors shielded from interaction with thereversed phase resin. Aggregated species are usually eluted at higheracetonitrile concentrations. In addition to resolving misfolded forms ofproteins from the desired form, the reversed phase step also removesendotoxin from the samples.

Fractions containing the desired folded PRO10282 or PRO19578 polypeptideare pooled and the acetonitrile removed using a gentle stream ofnitrogen directed at the solution. Proteins are formulated into 20 mMHepes, pH 6.8 with 0.14 M sodium chloride and 4% mannitol by dialysis orby gel filtration using G25 Superfine (Pharmacia) resins equilibrated inthe formulation buffer and sterile filtered.

Specifically, two extracellular domains (ECD) of the native human Stra6protein PRO10282, Peptide A (amino acids 229-295) and Peptide B (aminoacids 532-667) were expressed separately as peptides in the E. colicytoplasm with an N-terminal polyhistidine leader having the amino acidsequence MKHQHQHQHQHQHQMHQ (SEQ ID NO: 12). This leader provides foroptimal translation initiation, purification on a nickel chelationcolumn, and efficient removal if desired with the TAGZyme system(Unizyme Laboratories). Transcription was controlled by the E. colialkaline phosphatase promoter (Kikuchi et al., Nucleic Acids Res.9:5671-5678 [1981]) and the trp operon ribosome binding site (Yanofskyet al., Nucleic Acids Res. 9:6647-6668 [1981]) provided for translation.Downstream of the translation termination codon, is the λtotranscriptional terminator (Scholtissek and Grosse, Nucleic Acids Res.15:3185 [1987]) followed by the rare codon tRNA genes pro2, argU, andglyT (Komine et al., J. Mol. Biol. 212:579-598 [1990]; Fournier andOzeki, Microbiol. Rev. 49:379-397 [1985]).

The two Stra6 ECD coding sequence DNA fragments were prepared by PCRfrom a full length cDNA clone, and inserted into the expression vectordescribed above, which was designated as pST239. After DNA sequenceverification, the new Stra6 expression plasmids, designated PE148380Aand PE148380B, were transformed into the E. coli strain 58F3((fhuAΔ(tonAΔ) lonΔ galE rpoHts (htpRts) ΔclpP lacIq ΔompTΔ (nmpc-fepE)ΔslyD). Luria Broth cultures of these transformants were first grownovernight at 30° C., and then diluted 100 fold into a phosphate limitingmedia to induce the alkaline phosphatase promoter. After 24 hours at 30°C. with shaking, the cultures were centrifuged, and the cell pastesfrozen until the start of peptide purification.

For purification, E. coli pastes (6-10 gm pellets) were resuspended in10 volumes (w/v) of 7 M guanidine HCl, 20 mM Tris, pH 8, buffer. Solidsodium sulfite and sodium tetrathionate were added to make finalconcentrations of 0.1 M and 0.02 M, respectively, and the solution wasstirred overnight at 4° C. The solution was clarified by centrifugationand loaded onto a Qiagen Ni-NTA metal chelate column equilibrated in 6 Mguanidine, HCl, 20 mM Tris, pH 7.4. The column was washed withadditional buffer containing 50 mM imidazole (Calbiochem, Utrol grade).The protein was eluted with buffer containing 250 mM imidazole.Fractions containing the desired protein were pooled, dialyzed against 1mM HCl and stored at 4° C.

Example 4 Expression of PRO10282 and PRO19578 in Mammalian Cells

This example illustrates preparation of a potentially glycosylated formof PRO10282 and PRO19578 by recombinant expression in mammalian cells.

The vector, pRK5 (see EP 307,247, published Mar. 15, 1989), is employedas the expression vector. Optionally, the PRO10282 or PRO19578 DNA isligated into pRK5 with selected restriction enzymes to allow insertionof the PRO10282 DNA using ligation methods such as described in Sambrooket al., supra. The resulting vector is called pRK5 PRO10282 andpRK5-PRO19578, respectively.

In one embodiment, the selected host cells may be 293 cells. Human 293cells (ATCC CCL 1573) are grown to confluence in tissue culture platesin medium such as DMEM supplemented with fetal calf serum andoptionally, nutrient components and/or antibiotics. About 10 gpRK5-PRO10282 or pRK5-PRO19578 DNA is mixed with about 1 g DNA encodingthe VA RNA gene [Thimmappaya et al., Cell, 31:543 (1982)] and dissolvedin 500 l of 1 mM Tris-HCl, 0.1 mM EDTA, 0.227 M CaCl₂. To this mixtureis added, dropwise, 5001 of 50 mM HEPES (pH 7.35), 280 mM NaCl, 1.5 mMNaPO₄, and a precipitate is allowed to form for 10 minutes at 25° C. Theprecipitate is suspended and added to the 293 cells and allowed tosettle for about four hours at 37° C. The culture medium is aspiratedoff and 2 ml of 20% glycerol in PBS is added for 30 seconds. The 293cells are then washed with serum free medium, fresh medium is added andthe cells are incubated for about 5 days.

Approximately 24 hours after the transfections, the culture medium isremoved and replaced with culture medium (alone) or culture mediumcontaining 200 Ci/ml ³⁵S-cysteine and 200 Ci/ml ³⁵S-methionine. After a12 hour incubation, the conditioned medium is collected, concentrated ona spin filter, and loaded onto a 15% SDS gel. The processed gel may bedried and exposed to film for a selected period of time to reveal thepresence of PRO10282 polypeptide. The cultures containing transfectedcells may undergo further incubation (in serum free medium) and themedium is tested in selected bioassays.

In an alternative technique, PRO10282 or PRO19578 may be introduced into293 cells transiently using the dextran sulfate method described bySomparyrac et al., Proc. Natl. Acad. Sci., 12:7575 (1981). 293 cells aregrown to maximal density in a spinner flask and 700 g pRK5-PRO10282 orpRK5-PRO19578 DNA is added. The cells are first concentrated from thespinner flask by centrifugation and washed with PBS. The DNA-dextranprecipitate is incubated on the cell pellet for four hours. The cellsare treated with 20% glycerol for 90 seconds, washed with tissue culturemedium, and re-introduced into the spinner flask containing tissueculture medium, 5 g/ml bovine insulin and 0.1 g/ml bovine transferrin.After about four days, the conditioned media is centrifuged and filteredto remove cells and debris. The sample containing expressed PRO10282 orPRO19578 can then be concentrated and purified by any selected method,such as dialysis and/or column chromatography.

In another embodiment, PRO10282 or PRO19578 can be expressed in CHOcells. The pRK5-PRO10282 or pRK5-PRO19578 can be transfected into CHOcells using known reagents such as CaPO₄ or DEAE-dextran. As describedabove, the cell cultures can be incubated, and the medium replaced withculture medium (alone) or medium containing a radiolabel such as³⁵S-methionine. After determining the presence of PRO10282 or PRO19578polypeptide, the culture medium may be replaced with serum free medium.Preferably, the cultures are incubated for about 6 days, and then theconditioned medium is harvested. The medium containing the expressedPRO10282 or PRO19578 can then be concentrated and purified by anyselected method.

Epitope-tagged PRO10282 or pRO19578 may also be expressed in host CHOcells. The PRO10282 or PRO19578 may be subcloned out of the pRK5 vector.The subclone insert can undergo PCR to fuse in frame with a selectedepitope tag such as a poly-his tag into a Baculovirus expression vector.The poly-his tagged PRO10282 or PRO19578 insert can then be subclonedinto a SV40 driven vector containing a selection marker such as DHFR forselection of stable clones. Finally, the CHO cells can be transfected(as described above) with the SV40 driven vector. Labeling may beperformed, as described above, to verify expression. The culture mediumcontaining the expressed poly-His tagged PRO10282 or PRO19578 can thenbe concentrated and purified by any selected method, such as byNi²⁺-chelate affinity chromatography.

PRO10282 or PRO19578 may also be expressed in CHO and/or COS cells by atransient expression procedure or in CHO cells by another stableexpression procedure.

Stable expression in CHO cells is performed using the followingprocedure. The proteins are expressed as an IgG construct(immunoadhesin), in which the coding sequences for the soluble forms(e.g. extracellular domains) of the respective proteins are fused to anIgG1 constant region sequence containing the hinge, CH2 and CH2 domainsand/or is a poly-His tagged form.

Following PCR amplification, the respective DNAs are subcloned in a CHOexpression vector using standard techniques as described in Ausubel etal., Current Protocols of Molecular Biology, Unit 3.16, John Wiley andSons (1997). CHO expression vectors are constructed to have compatiblerestriction sites 5′ and 3′ of the DNA of interest to allow theconvenient shuttling of cDNA's. The vector used expression in CHO cellsis as described in Lucas et al., Nucl. Acids Res. 24:9 (1774-1779(1996), and uses the SV40 early promoter/enhancer to drive expression ofthe cDNA of interest and dihydrofolate reductase (DHFR). DHFR expressionpermits selection for stable maintenance of the plasmid followingtransfection.

Twelve micrograms of the desired plasmid DNA is introduced intoapproximately 10 million CHO cells using commercially availabletransfection reagents Superfect® (Quiagen), Dosper® or Fugene®(Boehringer Mannheim). The cells are grown as described in Lucas et al.,supra. Approximately 3×10⁻⁷ cells are frozen in an ampule for furthergrowth and production as described below.

The ampules containing the plasmid DNA are thawed by placement intowater bath and mixed by vortexing. The contents are pipetted into acentrifuge tube containing 10 mLs of media and centrifuged at 1000 rpmfor 5 minutes. The supernatant is aspirated and the cells areresuspended in 10 mL of selective media (0.2 μm filtered PS20 with 5%0.2 μm diafiltered fetal bovine serum). The cells are then aliquotedinto a 100 mL spinner containing 90 mL of selective media. After 1-2days, the cells are transferred into a 250 mL spinner filled with 150 mLselective growth medium and incubated at 37° C. After another 2-3 days,250 mL, 500 mL and 2000 mL spinners are seeded with 3×10⁵ cells/mL. Thecell media is exchanged with fresh media by centrifugation andresuspension in production medium. Although any suitable CHO media maybe employed, a production medium described in U.S. Pat. No. 5,122,469,issued Jun. 16, 1992 may actually be used. A 3 L production spinner isseeded at 1.2×10⁶ cells/mL. On day 0, the cell number pH is determined.On day 1, the spinner is sampled and sparging with filtered air iscommenced. On day 2, the spinner is sampled, the temperature shifted to33° C., and 30 mL of 500 g/L glucose and 0.6 μL of 10% antifoam (e.g.,35% polydimethylsiloxane emulsion, Dow Corning 365 Medical GradeEmulsion) taken. Throughout the production, the pH is adjusted asnecessary to keep it at around 7.2. After 10 days, or until theviability dropped below 70%, the cell culture is harvested bycentrifugation and filtering through a 0.22 μm filter. The filtrate waseither stored at 4° C. or immediately loaded onto columns forpurification.

For the poly-His tagged constructs, the proteins are purified using aNi-NTA column (Qiagen). Before purification, imidazole is added to theconditioned media to a concentration of 5 mM. The conditioned media ispumped onto a 6 ml Ni-NTA column equilibrated in 20 mM Hepes, pH 7.4,buffer containing 0.3 M NaCl and 5 mM imidazole at a flow rate of 4-5ml/min. at 4° C. After loading, the column is washed with additionalequilibration buffer and the protein eluted with equilibration buffercontaining 0.25 M imidazole. The highly purified protein is subsequentlydesalted into a storage buffer containing 10 mM Hepes, 0.14 M NaCl and4% mannitol, pH 6.8, with a 25 ml G25 Superfine (Pharmacia) column andstored at −80° C.

Immunoadhesin (Fc-containing) constructs are purified from theconditioned media as follows. The conditioned medium is pumped onto a 5ml Protein A column (Pharmacia) which had been equilibrated in 20 mM Naphosphate buffer, pH 6.8. After loading, the column is washedextensively with equilibration buffer before elution with 100 mM citricacid, pH 3.5. The eluted protein is immediately neutralized bycollecting 1 ml fractions into tubes containing 275 μL of 1 M Trisbuffer, pH 9. The highly purified protein is subsequently desalted intostorage buffer as described above for the poly-His tagged proteins. Thehomogeneity is assessed by SDS polyacrylamide gels and by N-terminalamino acid sequencing by Edman degradation.

Example 5 Expression of PRO10282 and PRO19578 in Yeast

The following method describes recombinant expression of PRO10282 andPRO19578 in yeast.

First, yeast expression vectors are constructed for intracellularproduction or secretion of PRO10282 polypeptides from the ADH2/GAPDHpromoter. DNA encoding a PRO10282 polypeptide (including PRO19578) andthe promoter is inserted into suitable restriction enzyme sites in theselected plasmid to direct intracellular expression of PRO10282. Forsecretion, DNA encoding PRO10282 can be cloned into the selectedplasmid, together with DNA encoding the ADH2/GAPDH promoter, a nativePRO10282 signal peptide or other mammalian signal peptide, or, forexample, a yeast alpha-factor or invertase secretory signal/leadersequence, and linker sequences (if needed) for expression of PRO10282.

Yeast cells, such as yeast strain AB110, can then be transformed withthe expression plasmids described above and cultured in selectedfermentation media. The transformed yeast supernatants can be analyzedby precipitation with 10% trichloroacetic acid and separation bySDS-PAGE, followed by staining of the gels with Coomassie Blue stain.

Recombinant PRO10282 (including PRO19578) can subsequently be isolatedand purified by removing the yeast cells from the fermentation medium bycentrifugation and then concentrating the medium using selectedcartridge filters. The concentrate containing PRO10282 (e.g. PRO19578)may further be purified using selected column chromatography resins.

Example 6 Expression of PRO10282 and PRO19578 in Baculovirus-InfectedInsect Cells

The following method describes recombinant expression of PRO10282 andPRO19578 in Baculovirus-infected insect cells.

The sequence coding for PRO10282 or PRO19578 is fused upstream of anepitope tag contained within a baculovirus expression vector. Suchepitope tags include poly-his tags and immunoglobulin tags (like Fcregions of IgG). A variety of plasmids may be employed, includingplasmids derived from commercially available plasmids such as pVL1393(Novagen). Briefly, the sequence encoding PRO10282 or PRO19578 or thedesired portion of the coding sequence of PRO10282 or PRO19578, such asthe sequence encoding the extracellular domain of a transmembraneprotein or the sequence encoding the mature protein if the protein isextracellular, is amplified by PCR with primers complementary to the 5′and 3′ regions. The 5′ primer may incorporate flanking (selected)restriction enzyme sites. The product is then digested with thoseselected restriction enzymes and subcloned into the expression vector.

Recombinant baculovirus is generated by co-transfecting the aboveplasmid and BaculoGold™ virus DNA (Pharmingen) into Spodopterafrugiperda (“Sf9”) cells (ATCC CRL 1711) using lipofectin (commerciallyavailable from GIBCO-BRL). After 4-5 days of incubation at 28° C., thereleased viruses are harvested and used for further amplifications.Viral infection and protein expression are performed as described byO'Reilley et al., Baculovirus expression vectors: A Laboratory Manual,Oxford: Oxford University Press (1994).

Expressed poly-his tagged PRO10282 or PRO19578 can then be purified, forexample, by Ni²⁺-chelate affinity chromatography as follows. Extractsare prepared from recombinant virus-infected Sf9 cells as described byRupert et al., Nature, 362:175-179 (1993). Briefly, Sf9 cells arewashed, resuspended in sonication buffer (25 mL Hepes, pH 7.9; 12.5 mMMgCl₂; 0.1 mM EDTA; 10% glycerol; 0.1% NP-40; 0.4 M KCl), and sonicatedtwice for 20 seconds on ice. The sonicates are cleared bycentrifugation, and the supernatant is diluted 50-fold in loading buffer(50 mM phosphate, 300 mM NaCl, 10% glycerol, pH 7.8) and filteredthrough a 0.45 μm filter. A Ni²⁺-NTA agarose column (commerciallyavailable from Qiagen) is prepared with a bed volume of 5 mL, washedwith 25 mL of water and equilibrated with 25 mL of loading buffer. Thefiltered cell extract is loaded onto the column at 0.5 mL per minute.The column is washed to baseline A₂₈₀ with loading buffer, at whichpoint fraction collection is started. Next, the column is washed with asecondary wash buffer (50 mM phosphate; 300 mM NaCl, 10% glycerol, pH6.0), which elutes nonspecifically bound protein. After reaching A₂₈₀baseline again, the column is developed with a 0 to 500 mM Imidazolegradient in the secondary wash buffer. One mL fractions are collectedand analyzed by SDS-PAGE and silver staining or Western blot withNi²⁺-NTA-conjugated to alkaline phosphatase (Qiagen). Fractionscontaining the eluted His₁₀-tagged PRO10282 or PRO19578 are pooled anddialyzed against loading buffer.

Alternatively, purification of the IgG tagged (or Fc tagged) PRO10282 orPRO19578 can be performed using known chromatography techniques,including for instance, Protein A or protein G column chromatography.

Example 7 Preparation of Antibodies that Bind PRO10282 or PRO19578

This example illustrates preparation of monoclonal antibodies which canspecifically bind PRO10282 or PRO19578.

Techniques for producing the monoclonal antibodies are known in the artand are described, for instance, in Goding, supra. Immunogens that maybe employed include purified PRO10282 and PRO19578, fusion proteinscontaining PRO10282 or PRO19578, and cells expressing recombinantPRO10282 or PRO19578 on the cell surface. Selection of the immunogen canbe made by the skilled artisan without undue experimentation.

Mice, such as Balb/c, are immunized with the PRO10282 or PRO19578immunogen emulsified in complete Freund's adjuvant and injectedsubcutaneously or intraperitoneally in an amount from 1-100 micrograms.Alternatively, the immunogen is emulsified in MPL-TDM adjuvant (RibiImmunochemical Research, Hamilton, Mont.) and injected into the animal'shind foot pads. The immunized mice are then boosted 10 to 12 days laterwith additional immunogen emulsified in the selected adjuvant.Thereafter, for several weeks, the mice may also be boosted withadditional immunization injections.

Serum samples may be periodically obtained from the mice byretro-orbital bleeding for testing in ELISA assays to detectanti-PRO10282 antibodies or anti-PRO19578 antibodies. After a suitableantibody titer has been detected, the animals “positive” for antibodiescan be injected with a final intravenous injection of PRO10282 orPRO19578. Three to four days later, the mice are sacrificed and thespleen cells are harvested. The spleen cells are then fused (using 35%polyethylene glycol) to a selected murine myeloma cell line such asP3X63AgU.1, available from ATCC, No. CRL 1597. The fusions generatehybridoma cells which can then be plated in 96 well tissue cultureplates containing HAT (hypoxanthine, aminopterin, and thymidine) mediumto inhibit proliferation of non-fused cells, myeloma hybrids, and spleencell hybrids.

The hybridoma cells will be screened in an ELISA for reactivity againstPRO10282 or PRO19578. Determination of “positive” hybridoma cellssecreting the desired monoclonal antibodies against PRO10282 or PRO19578is within the skill in the art.

The positive hybridoma cells can be injected intraperitoneally intosyngeneic Balb/c mice to produce ascites containing the anti-PRO10282 oranti-PRO19578 monoclonal antibodies. Alternatively, the hybridoma cellscan be grown in tissue culture flasks or roller bottles. Purification ofthe monoclonal antibodies produced in the ascites can be accomplishedusing ammonium sulfate precipitation, followed by gel exclusionchromatography. Alternatively, affinity chromatography based uponbinding of antibody to protein A or protein G can be employed.

Specifically, five Balb/c mice (Charles River Laboratories, Wilmington,Del.) were hyper-immunized with purified Unizyme-conjugated amino acidpeptide, corresponding to amino acids 532-667 of human Stra6, in Ribiadjuvant (Ribi Immunochem Research, Inc., Hamilton, Mo.). B-cells frompopliteal lymph nodes were fused with mouse myeloma cells (X63.Ag8.653;American Type Culture Collection, Rockville, Md.) as previouslydescribed (Hongo et al., Hybridoma 14:253-260 [1995]). After 10-14 days,supernatants were harvested and screened for antibody production bydirect enzyme-linked immunosorbant assay (ELISA). Eight positive clones,showing the highest immunobinding by direct ELISA andimmunohistochemistry after two rounds of subcloning by limitingdilution, were injected into Pristine-primed mice for in vivo productionof mAb (Freud and Blair; J. Immunol. 129:2826-2830 [1982]). The ascitesfluids were pooled and purified by Protein A affinity chromatography(Pharmacia fast protein liquid chromatography (FPLC); Pharmacia,Uppsala, Sweden) as previously described (Hongo et al., supra). Thepurified antibody preparations were sterile filtered (0.2-μm pore size;Nalgene, Rochester, N.Y.) and stored at 4° C. in phosphate bufferedsaline (PBS).

Example 8 Purification of PRO10282 and PRO19578 Polypeptides UsingSpecific Antibodies

Native or recombinant PRO10282 and PRO19578 polypeptides may be purifiedby a variety of standard techniques in the art of protein purification.For example, pro-PRO10282 or pro-PRO19578 polypeptide, mature PRO10282or PRO19578 polypeptide, or pre-PRO10282 or pre-PRO19578 polypeptide ispurified by immunoaffinity chromatography using antibodies specific forthe PRO10282 polypeptide of interest. In general, an immunoaffinitycolumn is constructed by covalently coupling the anti-PRO10282 oranti-PRO19578 polypeptide antibody to an activated chromatographicresin.

Polyclonal immunoglobulins are prepared from immune sera either byprecipitation with ammonium sulfate or by purification on immobilizedProtein A (Pharmacia LKB Biotechnology, Piscataway, N.J.). Likewise,monoclonal antibodies are prepared from mouse ascites fluid by ammoniumsulfate precipitation or chromatography on immobilized Protein A.Partially purified immunoglobulin is covalently attached to achromatographic resin such as CnBr-activated SEPHAROSE™ (Pharmacia LKBBiotechnology). The antibody is coupled to the resin, the resin isblocked, and the derivative resin is washed according to themanufacturer's instructions.

Such an immunoaffinity column is utilized in the purification ofPRO10282 or PRO19578 polypeptide by preparing a fraction from cellscontaining PRO10282 or PRO19578 polypeptide in a soluble form. Thispreparation is derived by solubilization of the whole cell or of asubcellular fraction obtained via differential centrifugation by theaddition of detergent or by other methods well known in the art.Alternatively, soluble PRO10282 or PRO19578 polypeptide containing asignal sequence may be secreted in useful quantity into the medium inwhich the cells are grown.

A soluble PRO10282 or PRO19578 polypeptide-containing preparation ispassed over the immunoaffinity column, and the column is washed underconditions that allow the preferential absorbance of PRO10282 orPRO19578 polypeptide (e.g., high ionic strength buffers in the presenceof detergent). Then, the column is eluted under conditions that disruptantibody/PRO10282 or PRO19578 polypeptide binding (e.g., a low pH buffersuch as approximately pH 2-3, or a high concentration of a chaotropesuch as urea or thiocyanate ion), and PRO10282 or pRO19578 polypeptideis collected.

Example 9 Drug Screening

This invention is particularly useful for screening compounds by usingPRO10282 (Stra6) polypeptides (including PRO19578) or binding fragmentthereof in any of a variety of drug screening techniques. The PRO10282polypeptide or fragment employed in such a test may either be free insolution, affixed to a solid support, borne on a cell surface, orlocated intracellularly. One method of drug screening utilizeseukaryotic or prokaryotic host cells which are stably transformed withrecombinant nucleic acids expressing the PRO10282 polypeptide orfragment. Drugs are screened against such transformed cells incompetitive binding assays. Such cells, either in viable or fixed form,can be used for standard binding assays. One may measure, for example,the formation of complexes between PRO10282 polypeptide or a fragmentand the agent being tested. Alternatively, one can examine thediminution in complex formation between the PRO10282 polypeptide and itstarget cell or target receptors caused by the agent being tested.

Thus, the present invention provides methods of screening for drugs orany other agents which can affect a PRO10282 (Stra6)polypeptide-associated disease or disorder. These methods comprisecontacting such an agent with a Stra6 polypeptide or fragment thereofand assaying (i) for the presence of a complex between the agent and theStra6 polypeptide or fragment, or (ii) for the presence of a complexbetween the Stra6 polypeptide or fragment and the cell, by methods wellknown in the art. In such competitive binding assays, the Stra6polypeptide or fragment is typically labeled. After suitable incubation,free Stra6 polypeptide or fragment is separated from that present inbound form, and the amount of free or uncomplexed label is a measure ofthe ability of the particular agent to bind to Stra6 polypeptide or tointerfere with the Stra6 polypeptide/cell complex.

Another technique for drug screening provides high throughput screeningfor compounds having suitable binding affinity to a polypeptide and isdescribed in detail in WO 84/03564, published on Sep. 13, 1984. Brieflystated, large numbers of different small peptide test compounds aresynthesized on a solid substrate, such as plastic pins or some othersurface. As applied to a PRO10282 (Stra6) polypeptide, the peptide testcompounds are reacted with Stra6 polypeptide and washed. Bound Stra6polypeptide is detected by methods well known in the art. Purified Stra6polypeptide can also be coated directly onto plates for use in theaforementioned drug screening techniques. In addition, non-neutralizingantibodies can be used to capture the peptide and immobilize it on thesolid support.

This invention also contemplates the use of competitive drug screeningassays in which neutralizing antibodies capable of binding a Stra6polypeptide specifically compete with a test compound for binding to aStra6 polypeptide or fragments thereof. In this manner, the antibodiescan be used to detect the presence of any peptide which shares one ormore antigenic determinants with the Stra6 polypeptide.

Example 10 Rational Drug Design

The goal of rational drug design is to produce structural analogs ofbiologically active polypeptide of interest (i.e., a PRO10282 (Stra6)polypeptide) or of small molecules with which they interact, e.g.,agonists, antagonists, or inhibitors. Any of these examples can be usedto fashion drugs which are more active or stable forms of the nativesequence PRO10282 polypeptide or PRO19578 polypeptide, or which enhanceor interfere with the function of the native sequence PRO10282 orPRO19578 polypeptide in vivo (c.f., Hodgson, Bio/Technology, 9: 19-21(1991)).

In one approach, the three-dimensional structure of the native sequencePRO10282 or PRO19578 polypeptide, or of a PRO10282 or PRO19578polypeptide-inhibitor complex, is determined by x-ray crystallography,by computer modeling or, most typically, by a combination of the twoapproaches. Both the shape and charges of the native PRO10282 orPRO19578 polypeptide must be ascertained to elucidate the structure andto determine active site(s) of the molecule. Less often, usefulinformation regarding the structure of the PRO10282 or PRO19578polypeptide may be gained by modeling based on the structure ofhomologous proteins. In both cases, relevant structural information isused to design analogous PRO10282 polypeptide-like molecules or toidentify efficient inhibitors. Useful examples of rational drug designmay include molecules which have improved activity or stability as shownby Braxton and Wells, Biochemistry, 31:7796-7801 (1992) or which act asinhibitors, agonists, or antagonists of native peptides as shown byAthauda et al., J. Biochem., 113:742-746 (1993).

It is also possible to isolate a target-specific antibody, selected byfunctional assay, as described above, and then to solve its crystalstructure. This approach, in principle, yields a pharmacore upon whichsubsequent drug design can be based. It is possible to bypass proteincrystallography altogether by generating anti-idiotypic antibodies(anti-ids) to a functional, pharmacologically active antibody. As amirror image of a mirror image, the binding site of the anti-ids wouldbe expected to be an analog of the original receptor. The anti-id couldthen be used to identify and isolate peptides from banks of chemicallyor biologically produced peptides. The isolated peptides would then actas the pharmacore.

By virtue of the present invention, sufficient amounts of the PRO10282polypeptides (including PRO19578) may be made available to perform suchanalytical studies as X-ray crystallography. In addition, knowledge ofthe PRO10282 polypeptide amino acid sequences provided herein willprovide guidance to those employing computer modeling techniques inplace of or in addition to x-ray crystallography.

Example 11 Tissue Expression Distribution

Oligonucleotide probes were constructed from the PRO10282polypeptide-encoding nucleotide sequence shown in the accompanyingfigures for use in quantitative PCR amplification reactions. Theoligonucleotide probes were chosen so as to give an approximately200-600 base pair amplified fragment from the 3′ end of its associatedtemplate in a standard PCR reaction. The oligonucleotide probes wereemployed in standard quantitative PCR amplification reactions withClontech RNA isolated from different adult human tissue sources andanalyzed by agarose gel electrophoresis so as to obtain a quantitativedetermination of the level of expression of the PRO10282polypeptide-encoding nucleic acid in the various tissues tested.Knowledge of the expression pattern or the differential expression ofthe PRO10282 polypeptide-encoding nucleic acid in various differenthuman tissue types provides a diagnostic marker useful for tissuetyping, with or without other tissue-specific markers, for determiningthe primary tissue source of a metastatic tumor, and the like. Theresults of these assays (shown in FIG. 10) demonstrated that the DNA148380-2827 molecule is highly expressed in the adult kidney, testis anduterus; significantly expressed in breast, prostate and trachea; weaklyexpressed in brain, heart, lung and thymus; and not expressed in liver,bone marrow, colon, skeletal muscle, small intestine, spleen andstomach.

Total RNA was purchased from Clontech (Palo Alto, Calif.) and analyzedusing the following primer/probe set for PCR amplification:

(SEQ ID NO: 6) h.Stra6.tmf3: 5′ CACACTCGAGAGCCAGATATTTT (SEQ ID NO: 7)h.Stra5.tmr4: 5′ AACAAGTTTATTGCAGGGAACAC (SEQ ID NO: 8) h.Stra6.tmp4:5′ TGTAGTTTTTATGCCTTTGGCTATTATGAAAGAGGT tmf = forward primer tmr= reverse primer tmp = probe

In situ hybridization, performed as described in Example 12 below,confirmed Stra6 expression in kidney tubular epithelial cells,myometrium, and stromal cells surrounding breast ducts and lobules,whereas little or no expression was detected in sections of brain,liver, spleen, pancreas, heart, lung, stomach, small intestine, colon,prostate, spleen, and adrenal cortex (data not shown). Of the normaltissues examined by in situ hybridization, highest expression levelswere seen in placenta and adrenal medulla, which were not included inthe PCR analysis.

Example 12 Over-Expression of Native Human PRO10282 (Stra6) Transcriptin Human Tumors

This example shows that the gene encoding native human full-lengthPRO10282 (Stra6) is significantly over-expressed in certain human colontumors and also in cell lines derived from various human tumors such ascolon, lung, kidney and breast.

The starting material for the screen was total RNA isolated from humancolon tumors, or various human colon, kidney, breast, or lung tumor celllines. In colon tumor tissue, Stra6 RNA expression was determinedrelative to RNA from normal colon tissue (mucosa) from the same patient.Stra6 RNA expression in various tumor cell lines was determined incomparison with various normal cell lines (i.e., normal colon, kidneyand lung cell lines).

Real-time quantitative PCR (RT-PCR, for example, TAQMAN ABI PRIZM 7700™Sequence Detection System™ [Perkin Elmer, Applied Biosystems Division,Foster City, Calif.]), was used to monitor quantitative differences inthe level of expression of the PRO10292 (Stra6) encoding gene(corresponding to DNA148380-2827) in normal cells and cells derived fromcertain cancers or cancer cell lines, using Taqman assay reagents. 50 μlRT-PCR reactions consisted of 5 μl 10× Taqman Buffer A, 300 mM of eachdNTF, 5 mM MgCl₂, 10 units of RNAse inhibitor, 12.5 units of MuLVReverse Transcriptase, 1.25 units of AmpliTaq Gold DNA Polymerase, 200nM probe, 500 nM primers and 100 ng RNA Reaction conditions consisted ofreverse transcription at 48° C. for 30 minutes, denaturation at 95° C.for 25 seconds and 65° C. for one minute. Reaction products wereanalyzed on 4-20% polyacrylamide gels (Novex).

Standard curves were used to determine relative levels of expression foreach gene of interest as well as the glyceraldehyde-3-phosphatedehydrogenase (GAPDH) housekeeping gene for each sample analyzed.Relative normalized units were obtained by dividing the gene of interestmRNA level by the GAPDH mRNA level. Relative normalized units werecompared between experimental sample and control to determine foldinduction.

The results were used to determine whether the mRNA encoding PRO10282 isover-expressed in any of the primary colon cancers or colon, kidney,breast, or lung cancer cell lines that were screened. The histology ofsome matched human normal and colon tumor samples used for the analysis(see FIGS. 11 and 12) is shown below:

Number Histology 850 Invasive adenocarcinoma; no necrosis; goodpreservation. 851 Invasive adenocarcinoma; minimal necrosis; goodcondition. 892 Invasive adenocarcinoma; minimal necrosis; goodcondition. 869 Invasive adenocarcinoma; minimal necrosis; goodcondition. 893 Normal mucosa - dysplasia - invasive adenocarcinoma;minor necrosis; good condition. 870 Adenocarcinoma - severe dysplasia;minimal necrosis; good condition. 871 Adenocarcinoma - dysplasia -normal mucosa; no necrosis; good condition. 848 Adenocarcinoma - appearsto be arising in villous adenoma; normal mucosa/submucosa; no necrosis,good condition. 872 Invasive adenocarcinoma; about 70% of tumor isnecrotic; overall good preservation. 778 Adenocarcinoma - unusuallypapillary morphology; normal/hyperplastic mucosa; minimal necrosis;acceptable preservation. 17 Moderately well-differentiatedadenocarcinoma. 18 Well-differentiated adenocarcinoma. 64 Moderatelywell-differentiated adenocarcinoma. 76 Moderately well-differentiatedadenocarcinoma.

Human lung cell lines include the normal human lung fibroblast celllines MRC5 (CCL-171) and IMR90 (CCL-186), the human lung carcinomaepithelial cell line A549 (SRCC768, CCL-185), the human epidermoid lungcarcinoma cell line Calu-1 (SRCC769; HTB-54), the human anaplasticcarcinoma cell line Calu-6 (SRCC770, HTB-56—probably lung), the humanepithelial cell line NCI-H441 (SRCC772; HTB-174) which was derived frompericardial fluid of a patient with papillary adenocarcinoma of thelung, and the human lung squamous cell carcinoma cell line SW900(SRCC775; HTB-59), all available from ATCC.

Colon cell lines include, for example, the normal colon fibroblast cellline CCD112Co (CRL-1541), the human colorectal adenocarcinoma cell lineCaCo-2 (HTB-37), the human colorectal adenocarcinoma cell line WiDr(CCL-218), the human colorectal carcinoma cell line HCT116 (CCL-247),the human colorectal adenocarcinoma cell line SK-Col (HTB-39), the humancolorectal adenocarcinoma cell line COLO320 (SRCC778; CCL-220), thehuman colorectal adenocarcinoma cell line HT29 (SRCC779; HTB-38), thehuman colorectal adenocarcinoma cell line SW403 (CCL-230), the humancolon cancer cell line NCI/HCC2998, and the human colorectaladenocarcinoma cell line Colo320DM (CCL-220), all available from ATCC orother public sources.

Human breast carcinoma cell lines include the human breastadenocarcinoma cell line MCF7 (SRCC766; HTB-22), and the human breastcancer cell line NCI/ADR-RES, both of which are publicly available.

Kidney lines include the 293 cell line (CRL-1573) which is transformedwith adenovirus 5 DNA. Two Wilm's tumor cell lines were also included inthe analysis, G401 (CRL-1441) and SK-NEP-1 (CRL-1573).

RNA Preparation:

RNA was prepared from the foregoing cultured cell lines. The isolationwas performed using purification kit, buffer set and protease fromQiagen, according to the manufacturer's instructions and the descriptionbelow. More specifically, total RNA from cells in culture was isolatedusing Qiagen RNeasy midi-columns. Total RNA from tissue samples wasisolated using RNA Stat-60 (Tel-Test). RNA prepared from tumor wasisolated by cesium chloride density gradient centrifugation.

Solid Human Tumor Sample Preparation and Lysis:

Tumor samples were weighed and placed into 50 ml conical tubes and heldon ice. Processing was limited to no more than 250 mg tissue perpreparation (1 tip/preparation). The protease solution was freshlyprepared by diluting into 6.25 ml cold ddH₂O to a final concentration of20 μg/ml and stored at 4° C. G2 buffer (20 ml) was prepared by dilutingDNAse A to a final concentration of 200 μg/ml (from 100 mg/ml stock).The tumor tissue was homogenized in 10 ml G2 buffer for 60 seconds usingthe large tip of the polytron in a laminar-flow TC hood in order toavoid inhalation of aerosols, and held at room temperature. Betweensamples, the polytron was cleaned by spinning at 2×30 seconds each in 2L, ddH₂O, followed by G2 buffer (50 ml). If tissue was still present onthe generator tip, the apparatus was disassembled and cleaned.

Qiagen protease (prepared as indicated above, 1.0 ml) was added,followed by vortexing and incubation at 50° C. for 3 hours. Theincubation and centrifugation were repeated until the lysates were clear(e.g., incubating additional 30-60 minutes, pelleting at 3000×g for 10minutes, 4° C.).

Quantitation

The results obtained from the real-time PCR analysis of RNA wereinitially expressed as delta CT units. One unit corresponds to one PCRcycle or approximately a 2-fold amplification relative to normal, twounits correspond to 4-fold, 3 units to 8-fold amplification and so on.The data is converted to fold difference and presented as such.Initially, reverse transcriptase was used to synthesize cDNA from 100 ngtotal RNA or polyA+ RNA using oligo(dT) as a primer. The resultant cDNAwas then used as a template for PCR. Quantitation was obtained usingprimers derived from the 3′-untranslated regions of the PRO10282encoding sequence and a TAQMAN™ fluorescent probe corresponding to therespective intervening sequences. Using the 3′ region tends to avoidcrossing intron-exon boundaries in the genomic DNA, an essentialrequirement for accurate assessment of RNA expression using this method.The sequences for the primers and probes (forward, reverse, and probe)using for the PRO10282 encoding gene amplification were as follows:

One set included the forward and reverse primers and probe described inExample 11 above as SEQ ID Nos: 6, 7 and 8, respectively.

Another set included:

hStra6.tmfl1: 5′ AGACCAGGTCCCACACTGA (SEQ ID NO: 9) hStra6.tmr1:5′ TTCATAATAGCCAAAGGCATAAAA, (SEQ ID NO: 10) and h.Stra6.tmp1:5′ CTGCCCACACTCGAGAGCCAGAT 3′ (SEQ ID NO: 11)

Human GAPDH:

forward primer: 5′-GAAGATGGTGATGGGATTTC-3′ (SEQ ID NO: 14) reverseprimer; 5′-GAAGGTGAAGGTCGGAGTC-3′ (SEQ ID NO: 15) probe:5′-CAAGCTTCCCGTTCTCAGCC-3′ (SEQ ID NO: 16)

The 5′ nuclease assay reaction is a fluorescent PCR-based techniquewhich makes use of the 5′ exonuclease activity of Taq DNA polymeraseenzyme to monitor amplification in real time. Two oligonucleotideprimers are used to generate an amplicon typical of a PCR reaction. Athird oligonucleotide, or probe, is designed to detect nucleotidesequence located between the two PCR primers. The probe isnon-extendible by Taq DNA polymerase enzyme, and is labeled with areporter fluorescent dye and a quencher fluorescent dye. Anylaser-induced emission from the reporter dye is quenched by thequenching dye when the two dyes are located close together as they areon the probe. During the amplification reaction, the Taq DNA polymeraseenzyme cleaves the probe in a template-dependent manner. The resultantprobe fragments disassociate in solution, and signal from the releasedreporter dye is free from the quenching effect of the secondfluorophore. One molecule of reporter dye is liberated for each newmolecule synthesized, and detection of the unquenched reporter dyeprovides the basis for quantitative interpretation of the data.

As noted above, the 5′ nuclease procedure is run on a real-timequantitative PCR device such as the ABI PRIZM 7700™ Sequence DetectionSystem™. The system consists of a thermocycler, laser, charge-coupleddevice (CCD), camera and computer. The system amplifies samples in a96-well format on a thermocycler. During amplification, laser-inducedfluorescent signal is collected in real-time through fiber optics cablesfor all 96 wells, and detected at the CCD. The system includes softwarefor running the instrument and for analyzing the data.

5′-Nuclease assay data are initially expressed as Ct, or the thresholdcycle. This is defined as the cycle at which the reporter signalaccumulates above the background level of fluorescence. The Ct valuesare used as quantitative measurement of the relative number of startingcopies of a particular target sequence in a nucleic acid sample whencomparing the expression of RNA in a cancer cell with that from a normalcell.

Results

The human Stra6 RNA (corresponding to DNA148380-2827) shows strongover-expression in human colon tumor tissues, when compared withcorresponding normal human colon tissues. As shown in FIG. 11, humanStra6 RNA (corresponding to DNA 148380-2827) was found to beover-expressed in all 14 human tumor colon tissues examined, relative toRNA from matched normal colorectal mucosa from the same patient. Theover-expression varied between two- and 170-fold, and in 7 out of 14tumor tissue samples was at least about 10-fold. The cycle thresholdvalues obtained by quantitative PCR indicated that Stra6 mRNA levelswere extremely low or possibly absent in many of the normal mucosasamples.

As a second method of detection, the products obtained after completionof the quantitative PCR reactions (40 cycles each) were subjected toelectrophoresis in polyacrylamide gels and visualized by ethidiumbromide staining. As shown in FIG. 12A, using expression of ahousekeeping gene, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) asthe standard, substantially greater amounts of PCR products weregenerated off Stra6 mRNA in tumor samples compared to their normalcounterparts. By contrast, a comparable level of product from theinternal control GAPDH mRNA was generated from all samples.

Stra6 expression in colon adenocarcinomas was localized to theepithelial tumor cells by in situ hybridization (ISH) performed asfollows. ³²P-labeled sense and antisense riboprobes were transcribedfrom an 874 bp PCR product corresponding to nucleotides 432-1247 of thecoding sequence of human Stra6 polypeptide encoded by DNA148380-2827.Formalin-fixed, paraffin-embedded tissue sections were processes asdescribed previously (Pennica et al., Proc. Natl. Acad. Sci. USA95:14717-22 [1998]). The results are shown in FIG. 12B.

As shown in FIG. 13, the human Stra6 RNA (corresponding to DNA148380-2827) is also significantly over-expressed in various breast,kidney, colon and lung tumor cell lines. “Relative RNA Expression” meansthat the RNA expression in normal and tumor tissues is shown relative toexpression in an arbitrarily chosen standard cell line SW480.

In situ hybridization results obtained in various tumor sections arealso shown in FIG. 16. Several tumor types other than colonadenocarcinomas also showed high levels of Stra6 expression. Theseincluded 3 of 3 melanomas (FIGS. 16A and B), 3 of 4 endometrialadenocarcinomas (FIGS. 16C and D), 2 of 3 ovarian adenocarcinomas, and aWilm's tumor of the kidney (FIGS. 16E and F). The Stra6 in situhybridization signal in these various tumors was considerably greaterthan in colon adenocarcinomas consistent with data showing relativelyhigh expression levels in normal kidney and uterus and low levels innormal colon. Since Stra6 was detected in normal adrenal medulla, wealso examined two pheochromocytomas, which are tumors derived from thistissue. In these tumors, Stra6 expression exceeded that of any othertumor or tissue examined in this study (FIGS. 16G and H). Although Stra6was detected in normal kidney and was strongly expressed in Wilm'stumor, it was not detected in renal cell carcinomas. In kidneytransitional cell carcinomas, tumor-associated stromal cells rather thantumor epithelial cells expressed Stra6 (data not shown).

Because mRNA DNA148380-2827 encoding PRO10282 is overexpressed invarious tumors as well as in a number of tumor derived cell lines, it islikely associated with tumor formation and/or growth. As a result,antagonists (e.g., antibodies, organic and inorganic small molecules,peptides and polypeptides, such as Stra6 variants, antisenseoligonucleotides) directed against the protein encoded by DNA148380-2827(PRO10282) or other naturally occurring variants of this protein, suchas PRO19578 encoded by DNA148389-2827-1, are expected to be useful indiagnosis, prevention and/or treatment of cancer particularly, withoutlimitation, colon, lung, breast and/or kidney cancer.

The efficacy of antagonists such as therapeutic antibodies directedagainst the protein encoded by DNA148380-2827 (PRO10282) could beenhanced by agents that stimulate the expression of the gene encodingPRO10282. For example, treatment of human colorectal cancer cell lineswith 9-cis-retinoic acid or all-trans retinoic acid resulted in adramatic enhancement of the expression of the Stra6 mRNA (FIG. 15).Thus, the treatment of cancer patients with therapeutic antibodiesdirected against PRO10282 in combination with the appropriate retinoidswould be expected to enhance tumor killing by the antibodies. The samewill be true to antibodies directed against other native Stra6polypeptides over-expressed in various tumors, such as the human splicevariant encoded by DNA148389-2827-1.

Example 13 Synergistic Induction of Stra6 by Wnt-1 and RetinoidsMaterials and Methods

Cell Culture

C57MG and C57MG/Wnt-1 cells were grown in Dulbecco's Modified Eaglemedium supplemented with 10% fetal bovine serum, 2 mM L-glutamine, and2.5 μg/ml puromycin (Edge Biosystems). C57MG cells withtetracycline-repressible Wnt-1 expression were grown in complete mediumwithout puromycin, supplemented with 400 μg/ml G418 (Gibco BRL), 100μg/ml hygromycin B (Gibco BRL), and 50 ng/ml tetracycline (Korinek etal., Mol. Cell. Biol. 18:1248-56 [1998]). For Wnt-1 induction studies,cells were washed with phosphate buffered saline, cultured intetracycline-free media for 10, 24, 48, 72, and 96 hours and thenharvested. A O-hour control dish was maintained entirely in mediacontaining tetracycline. All dishes were simultaneously harvested andtotal RNA was extracted for each time point. RT-PCR was carried out withWnt-1, Stra6, and GAPDH specific primers and probes on 100 ng total RNAfrom each sample.

Human colon adenocarcinoma cell lines HCT116 and WiDr cells wereobtained from the American Type Culture Collection. HCT116 cells weremaintained in McCoy's 5A media supplemented with 10% fetal bovine serum(FBS). WiDr cells were maintained in Dulbecco's minimal essential media(DMEM) supplemented with 10% FBS. For retinoic acid induction studies,cells were plated at 10⁵ cells/60 mm dish containing 2.5 ml of mediumand allowed to grow for 24 hours. Cells were treated with Vitamin D3,all-trans-RA (Spectrum Laboratory Products), or 9-cis-RA (TorontoResearch Chemicals Inc.) (1 μM final concentration in DMSO) for theindicated times. Control cells were treated with an equal volume ofDMSO.

For the treatment of C57MG/Parent cells with Wnt-3a conditioned media,cells were incubated in regular media or conditioned media from L-cellsor L-W3A cells in the presence or absence of 1 μM 9-cis-RA. Conditionedmedia was prepared as previously described (Willert et al., Genes Dev.13:1768-73 [1999]). At 48 hours, cells were harvested by scraping intoPBS containing sodium fluoride and vanadate and quick frozen in liquidnitrogen. RNA was prepared using the RNAeasy kit (Qiagen), including theadditional DNaseI treatment on the columns according to manufacturer'sinstructions.

Western Blotting

For the C57MG/Wnt-1 TET cell line, Wnt-1 expression was induced byculturing cells in the absence of tetracycline for 0, 24, 48, or 72hours. Cells were lysed in Triton X-100 lysis buffer [20 mM tris-HCl (pH8.0), 137 mM NaCl, 1% Triton X-100, 1 mM EGTA, 10% glycerol, 1.5 mMMgCl₂, 1 mM dithiothreitol, 1 mM sodium vanadate, 50 mM sodium fluoride,and complete protease inhibitor cocktail (Boehringer Mannheim) andprotein-equivalents were subjected to SDS-PAGE and immunoblotting. Blotswere incubated with either 0.2 μg/ml affinity purified rabbit polyclonalantibody against β-catenin (Rubinfeld et al., Science 262:1731-1734[1993]), 0.1 μg/ml anti-ERK2 monoclonal antibody (TransductionLaboratories), or 1:2000 rabbit polyclonal antisera against RARγ-1(Affinity Bioreagents). For the WiDr cell line, cells were treated with1 μM all-trans-RA for 48 hours and then lysed in Triton X-100 lysisbuffer and processed as indicated above. Blots were incubated with 1:50anti-Stra6 peptide B monoclonal hybridoma culture supernatant (clone12F4.2H9.1D5).

Immunohistochemistry

WiDr cells were treated with 9-cis-retinoic acid or DMSO, then detachedand pelleted by low-speed centrifugation. Cell pellets were fixedovernight in 10% neutral buffered formalin, dehydrated, and embedded inparaffin. Immunohistochemistry was performed using anti-Stra6 peptide Bmonoclonal hybridoma culture supernatant (clone 12F4.2H9.1D5) ornonspecific mouse isotype IgG2A as primary antibodies, followed bydetection using avidin-biotin complex method with diaminobenzidine aschromogen (Vectastain Elite Kit, Vector Laboratories) as describedpreviously (Eberhard et al., Am. J. Pathol. 145:640-9 [1994]). Sectionswere counterstained with hematoxylin.

Wnt-1 Transgenic Mice

Transgenic mice that express the Wnt-1 proto-oncogene in the mammarygland typically exhibit hyperplastic lesions and develop neoplasms inthis tissue (Tsukamoto et al., Cell 55:619-625 [1988]). Such mice wereused in the following experiments.

Results

Easwaran et al. previously reported enhanced activation of a syntheticretinoic acid responsive reporter gene when MCF-7 cells wereco-transfected with mutant β-catenin and treated with retinoids(Easwaran et al., Curr. Biol. 9:1415-1418 [1999]). Considering this,together with the original identification of Stra6 as a retinoic acidinducible gene (Bouillet et al., Mech Dev. 63:173-186 [1997]), we askedwhether retinoic acid could synergize with Wnt-1 to increase the levelof Stra6 in the C57MG cell line. Treatment of parental C57MG cells witheither 9-cis-RA or all-trans-RA (ATRA) for 48 hours significantlyincreased the level of Stra6 mRNA while DMSO and vitamin D3 had noeffect (FIG. 17A). As expected, the C57MG/Wnt-1 cells treated witheither DMSO or vitamin D3 exhibited enhanced levels of Stra6 mRNArelative to the parent cell line. The level of Stra6 induction by Wnt-1was comparable to that observed on stimulation of the parental C57MGcells with 9-cis-RA. However, 9-cis-RA treatment of the C57MG/Wnt-1 cellline induced a further 10-fold increase in Stra6 mRNA relative to eitheruntreated C57MG/Wnt-1 or 9-cis-RA treated C57MG parent cells. Similarresults were obtained with all-trans-RA.

It was possible that potential clonal variations in the C57MG/Wnt-1 cellline, that were unrelated to Wnt-1 expression, accounted for theirdifferential response to retinoic acid relative to parental controlcells. To address this, we tested the response of the parental C57MGcells to stimulation by soluble Wnt-3a in the presence or absence of9-cis-RA. Wnt-3a is a Wnt-1 homolog that exhibits transformingproperties similar to those of Wnt-1 and can be produced as a solubleligand in mouse L-cells. Conditioned media from cultured L-cellsexpressing Wnt-3a, but not from control L-cells, induced the expressionof Stra6 in the C57MG cells (FIG. 17B). A slightly higher level ofinduction was observed on treatment of C57MG cells with 9-cis-RA.However, the combination of 9-cis-RA and Wnt-3a resulted in levels ofStra6 transcript vastly exceeding that seen with either agent alone.

If the induction of Stra6 in the retinoic acid treated C57MG/Wnt-1 cellswas potentiated by increased β-catenin levels, then one might expect asimilar induction of Stra6 in response to retinoic acid in human coloncarcinoma cells containing mutations in either β-catenin or APC. Todetermine whether this occurs, Stra6 mRNA levels were measured beforeand after retinoic acid treatment in HCT116 cells, which carry anactivating mutation in β-catenin, and in WiDr cells, which have lostboth copies of wild-type APC. In both cell lines, a significant increasein Stra6 mRNA levels was seen following treatment with either ATRA or9-cis-RA compared to DMSO or vitamin D3 (FIG. 17C). The activation ofthe Stra6 gene by ATRA in the HCT116 cell line was confirmed by in situhybridization (FIG. 17D). Induction of the predicted 73 kDa Stra6protein band in WiDr cells treated with ATRA was detected by Westernblot analysis with a Stra6 specific monoclonal antibody (FIG. 17E).Immunohistochemical analysis of the WiDr cells revealed that theincrease in Stra6 protein in response to retinoic acid was localized tothe plasma membrane (FIG. 17F).

The RARγ gene has been proposed as a target for Wnt signaling in Xenopusembryos, and the induction of Stra6 by retinoids was shown to bedependent upon the presence of this retinoic acid receptor subtype(McGrew et al., Mech. Dev. 87:21-32 [1999]; Taneja et al., Proc. Natl.Acad. Sci. USA 92:7854-8 [1995]). Together, these observations suggestthat the synergistic activation of Stra6 by Wnt and retinoids might bedue to the activation of RARγ expression by Wnt signaling. To determinewhether Wnt-1 signaling had any influence over the levels of RARγ inmammalian cells, we performed Western blots for the receptor on lysatesprepared from C57MG cells that conditionally express Wnt-1. Upon Wnt-1expression, a protein reactive with antibody specific to RARγ-1, andmigrating with an apparent molecular mass of 64 kDa, was induced at 24hours and its expression was increased at 48 hours (FIG. 18A). We alsoanalyzed hyperplastic mammary glands and mammary gland tumors obtainedfrom Wnt-1 transgenic mice and detected elevated levels of RARγ mRNA inthese tissues relative to normal mammary gland (FIG. 18B). The level ofRARγ transcript present in equivalent amounts of RNA isolated from 19adenocarcinomas was assessed by quantitative PCR. Notably, RARγ mRNAexpression was increased approximately 2-4 fold in 14 of the 19 (74%) ofhuman colon tumors examined compared to normal human colon tissue (FIG.18C). These results demonstrate that Wnt-1 signaling promotes theexpression of RARγ that the receptors are elevated in mouse and humantumors that are driven by the Wnt-1 pathway.

Discussion of Experimental Findings

Gene expression profiling approaches are based on unbiased detection ofmRNA transcripts and can therefore lead to unexpected insights into themechanisms by which gene activation occurs. Here it has been shown thatWnt-1 promoted the induction of the retinoic acid responsive gene Stra6,suggesting a connection between signaling pathways elicited by Wnt andretinoic acid. This connection was further supported by demonstrating asynergistic induction of Stra6 by a combination of Wnt and retinoicacid. There are at least three alternative explanations that couldaccount for this synergy: i) transcription factors directly responsiveto Wnt such as the TCF/LEFs might bind to and activate promoter elementsin the Stra6 gene; ii) signaling components in the Wnt pathway mightdirectly interact with the appropriate retinoic acid receptor (RAR) andpotentiate gene activation mediated by the RAR; or iii) signaling byWnt-1 could activate expression of the appropriate RAR, which could thensensitize cells to retinoic acid. Although this final proposal isfavored because the data presented herein show that Wnt-1 signalingrapidly induces expression of the RARγ receptor, the present inventionis not limited by any particular theory or mechanism of action.

Previous work has shown that specific disruption of the RARγ genegreatly reduced induction of the Stra6 gene by retinoids, which was thenrescued on re-expression of this receptor (Taneja et al., supra).However, it remains possible that mechanisms in addition to theWnt-1-induced expression of RARγ may also contribute to the observedsynergy. The proposal that β-catenin binds to retinoic acid receptors isattractive, but we have not observed any specific association ofendogenous RARγ with β-catenin in our cell lines. However, the bindingof β-catenin to RARγ, as was shown in vitro (Easwaran et al., 1999,supra), might relate to the activation of Stra6 by Wnt-1. The expressionpattern of Stra6 in transgenic animals null for RARγ was more widespreadthan that observed in wild-type littermates, and induction of Stra6 byretinoic acid was enhanced in cells from the null animals compared tocontrols (Bouillet et al., 1997, supra; Taneja et al., 1995, supra).Thus RARγ might be inhibitory to Stra6 expression, and activation ofβ-catenin by Wnt-1 could potentially relieve this inhibition ifβ-catenin inhibited RARγ function.

The synergistic induction of a retinoic acid responsive gene by Wnt-1signaling implies that human cancers that harbor genetic defects in theWnt-1 pathway would exhibit overexpression of Stra6. The vast majorityof colorectal tumors contain mutations in the genes coding for eitherthe APC tumor suppressor or β-catenin (Polakis, Curr. Opin. Genet. Dev.9:15-21 [1999]) and, accordingly, we have detected overexpression of theStra6 transcript in 14 of 14 colorectal tumors relative to matchednormal tissue (see Example 12). Activating mutations in β-catenin havealso been identified in cancers of the ovary and endometrium, Wilm'skidney tumors and melanomas, demonstrating that defects in Wnt-1signaling contribute to the progression of these cancers (Kobayashi etal. Jpn. J. Cancer Res. 90:55-9 [1999]; Koesters et al., Cancer Res.59:3880-2 [1999]; Palacios and Hamallo, Cancer Res. 58:1344-7 [1998];Rimm et al. Am. J. Pathol. 154:325-9 [1999]; Rubinfeld et al. Science262:1731-1734 [1993]; Wright et al. Int. J. Cancer 82:625-9 [1999]). Allfour of these human cancers displayed overexpression of Stra6 mRNA asdetermined by in situ hybridization (see Example 12). Pheochromocytoma,a tumor derived from the adrenal medulla, exhibited extremely highlevels of Stra6 mRNA, however, the status of these tumors with respectto mutations in the Wnt-1 signaling pathway has not been reported. It isintriguing that the cell types that give rise to melanomas andpheochromocytomas share in common an embryological derivation from theneural crest. Notably, Wilms' tumor of the kidney, pheochromocytoma, andendometrial carcinomas all arise in organs in which Stra6 is normallyexpressed. This suggests that tumorigenic signaling, such as that drivenby the Wnt-1 pathway, might interact with signals responsible fordifferentiation, thereby hyper-activating the expression of Stra6.

The human Stra6 protein resides at the cell surface, as determined bythe staining of colorectal cancer cells with monoclonal antibodiesspecific to human Stra6 protein. Moreover, the staining intensityobserved at the cell membrane was increased after treatment of cellswith retinoic acid. Thus, Stra6 likely encodes a multi-passtransmembrane protein that is localized to the cell surface.

Deposit of Material

The following materials have been deposited with the American TypeCulture Collection, 10801 University Blvd., Manassas, Va. 20110-2209,USA (ATCC):

Material ATCC Dep. No. Deposit Date DNA148380-2827 PTA-1181 Jan. 11,2000 DNA148389-2827-1 PTA-1402 Feb. 23, 2000

These deposits were made under the provisions of the Budapest Treaty onthe International Recognition of the Deposit of Microorganisms for thePurpose of Patent Procedure and the Regulations thereunder (BudapestTreaty). This assures maintenance of a viable culture of the deposit for30 years from the date of deposit. The deposit will be made available byATCC under the terms of the Budapest Treaty, and subject to an agreementbetween Genentech, Inc. and ATCC, which assures permanent andunrestricted availability of the progeny of the culture of the depositto the public upon issuance of the pertinent U.S. patent or upon layingopen to the public of any U.S. or foreign patent application, whichevercomes first, and assures availability of the progeny to one determinedby the U.S. Commissioner of Patents and Trademarks to be entitledthereto according to 35 U.S.C. § 122 and the Commissioner's rulespursuant thereto (including 37 C.F.R. § 1.14 with particular referenceto 886 OG 638).

The assignee of the present application has agreed that if a culture ofthe materials on deposit should die or be lost or destroyed whencultivated under suitable conditions, the materials will be promptlyreplaced on notification with another of the same. Availability of thedeposited material is not to be construed as a license to practice theinvention in contravention of the rights granted under the authority ofany government in accordance with its patent laws.

The foregoing written specification is considered to be sufficient toenable one skilled in the art to practice the invention. The presentinvention is not to be limited in scope by the construct deposited,since the deposited embodiment is intended as a single illustration ofcertain aspects of the invention and any constructs that arefunctionally equivalent are within the scope of this invention. Thedeposit of material herein does not constitute an admission that thewritten description herein contained is inadequate to enable thepractice of any aspect of the invention, including the best modethereof, nor is it to be construed as limiting the scope of the claimsto the specific illustrations that it represents. Indeed, variousmodifications of the invention in addition to those shown and describedherein will become apparent to those skilled in the art from theforegoing description and fall within the scope of the appended claims.

1-33. (canceled)
 34. An antibody or fragment thereof which specificallybinds to a Stra6 polypeptide.
 35. The antibody or antibody fragment ofclaim 34, which induces the death of a cell that expresses saidpolypeptide.
 36. The antibody or antibody fragment of claim 35, whereinsaid cell is a cancer cell that over-expresses said polypeptide ascompared to a normal cell of the same tissue type.
 37. The antibody orantibody fragment of claim 34 which is a monoclonal antibody or afragment of the monoclonal antibody.
 38. The antibody or antibodyfragment of claim 37 which comprises a non-human complementaritydetermining region (CDR) or a human framework region (FR).
 39. Theantibody or antibody fragment of claim 34 which is labeled.
 40. Theantibody or antibody fragment of claim 34 which is a single-chainantibody, a diabody, a linear antibody, or a fragment thereof.
 41. Theantibody or antibody fragment of claim 34 which is a humanized antibodyor a fragment of the humanized antibody.
 42. A composition of matterwhich comprises an antibody or antibody fragment of claim 34 inadmixture with a pharmaceutically acceptable carrier.
 43. Thecomposition of matter of claim 42, which comprises a therapeuticallyeffective amount of said antibody or antibody fragment.
 44. Thecomposition of matter of claim 42, which further comprises a cytotoxicor chemotherapeutic agent.
 45. An isolated nucleic acid molecule thatencodes the antibody or antibody fragment of claim
 34. 46. A vectorcomprising the nucleic acid molecule of claim
 45. 47. A host cellcomprising the vector of claim
 46. 48. A method for producing anantibody or antibody fragment that binds to a Stra6 polypeptide, saidmethod comprising culturing the host cell of claim 47 under conditionssufficient to allow expression of said antibody and recovering saidantibody or antibody fragment from the cell culture. 49-67. (canceled)68. A method for inhibiting the growth of tumor cells, said methodcomprising exposing tumor cells that express a Stra6 polypeptide to aneffective amount of the antibody or antibody fragment of claim 34,wherein growth of said tumor cells is thereby inhibited.
 69. The methodof claim 68, wherein said tumor cells over-express said Stra6polypeptide as compared to normal cells of the same tissue type. 70.(canceled)
 71. The method of claim 68, wherein said antibody or antibodyfragment induces tumor cell death.
 72. The method of claim 68, whereinsaid tumor cells are further exposed to radiation treatment, a cytotoxicagent, or a chemotherapeutic agent.
 73. The method of claim 68, whereinsaid tumor cells are further exposed to an agent that stimulates theexpression of the gene encoding Stra6.
 74. The method of claim 73wherein said agent is retinoic acid or an analogue thereof. 75-78.(canceled)
 79. An article of manufacture, comprising: a container; alabel on the container; and a composition comprising an antibody orantibody fragment contained within the container, wherein thecomposition is effective for inhibiting the growth of tumor cells andwherein the label on the container indicates that the composition iseffective for treating conditions characterized by over-expression of aStra6 polypeptide in said tumor cells as compared to normal cells of thesame tissue type.
 80. The article of manufacture of claim 79, whereinsaid antibody or antibody fragment inhibits a biological activity ofand/or the expression of said Stra6 polypeptide. 81-95. (canceled) 96.The antibody or antibody fragment of claim 34, wherein the antibodyfragment comprises a Fab, Fab′F (ab′)₂, or Fv fragment.